Novel expression vectors containing accessory molecule ligand genes and their use for immunomodulation and treatment of malignancies and autoimmune disease

ABSTRACT

This invention relates to genes which encode accessory molecule ligands and their use for immunomodulation, vaccination and treatments of various human diseases, including malignancies and autoimmune diseases. This invention also describes the use of accessory molecule ligands which are made up of various domains and subdomain portions of molecules derived from the tumor necrosis factor family. The chimeric molecules of this invention contain unique properties which lead to the stabilization of their activities and thus greater usefulness in the treatment of diseases. Vectors for expressing genes which encode the accessory molecule ligands of this invention are also disclosed.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.08/982,272 filed Dec. 1, 1997, which claims priority to U.S. Provisional60/132,145 filed Dec. 9, 1996, both of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel expression vectors containinggenes which encode an accessory molecule ligand and the use of thosevectors for immunomodulation, improved vaccination protocols and thetreatment of malignancies and autoimmune disease. More particularly,this invention provides expression vectors and methods for treatingvarious neoplastic or malignant cells, and expression vectors andmethods for treating autoimmune Disease. This invention alsocontemplates the production and expression of accessory molecule ligandswith greater stability and enhanced function.

BACKGROUND OF THE INVENTION

Leukemias, lymphomas, carcinomas and other malignancies are well knownand described in, e.g., Harrison's Principles of Internal Medicine,Wilson et al., eds., McGraw-Hill, New York, pp. 1599-1612. Thesemalignancies appear to have somehow escaped the immune systemsurveillance mechanisms that eliminate rapidly and continuouslyproliferating cells. The exact mechanism by which these malignanciesescape the immune system surveillance is not known.

Some of these malignant immune system cells are malignant antigenpresenting cells which do not function properly within the immunecascade. For example, neoplastic B cells cannot induce even weakallogeneic or autologous mixed lymphocyte reactions in vitro. Furtherevidence that malignancies survive due to the failure of the immunesurveillance mechanism includes the increased frequency of suchmalignancies in immunocompromised individuals, such as allograftrecipients and those receiving long-term immunosuppressant therapy.Further, the frequency of these malignancies is increased in patientshaving Acquired Immune Deficiency Syndrome (AIDS) and patients withprimary immune deficiency syndromes, such as X-linkedlymphoproliferative syndrome or Wiscott-Aldrich Syndrome (Thomas et al.,Adv. Cancer Res. 57:329, 1991).

The immune system normally functions to eliminate malignant cells byrecognizing the malignant cells as foreign cells and clearing thosecells from the body. An immune reaction depends on both the immunesystem's antibody response and on the cellular immune response within apatient. More specifically, the cellular immune response which acts torecognize the malignant cells as foreign requires a number of differentcells of the immune system and the interaction between those cells. Animmune reaction begins with a T lymphocyte (T cell) which has on itscell surface the T cell receptor. The T cell also has the ability toexpress on its surface various accessory molecules which interact withaccessory molecules on the B lymphocyte (B cell). When the T cellreceptor of the T cell specifically binds to a foreign antigen, such asa malignant cell, it becomes activated and expresses the accessorymolecule ligand, CD40 ligand on its cell surface. The accessory cellmolecule ligand is only present on the activated T cells for a shortperiod of time and is rapidly removed from the cell surface. After theaccessory cells molecule ligand is removed from the surface of theactivated T cell, its ability to bind to B cells via the accessorymolecule ligand is destroyed.

When present on the surface of an activated T cell, the accessory cellligand can specifically bind to the accessory cell molecule present onthe B cell. This specific T-B cell interaction causes the B and T cellto express costimulutory surface accessory molecule and cytokines whichresult in an immune activation which lead to cytolytic T cells whichspecifically kill and remove the malignant cell from the body.

The interaction with an activated T cell is not solely limited to Bcells but rather can be carried out by any cell which is able to presentantigen to the T cell (an antigen presenting cell). These cells includeB lymphocyte, macrophages, dendritic cells, monocytes, Langerhans cells,interdigitating cells, follicular dendritic cells or Kupffer cells.These cells all are known to have various accessory molecules on thecell surface which allow them to interact with other cells of the immunesystem. For example, these antigen presenting cells all have theaccessory molecule CD40 on their cell surface. The presence of theseaccessory molecules allows these antigen presenting cells tospecifically bind to complimentary accessory molecule ligand and thusdirectly interact with other immune cells.

A large number of accessory molecule ligands are members of the tumornecrosis factor superfamily. (Fanslow et al., Sem. Immun., 6:267-268(1994). The genes for a number of these accessory molecule ligands havebeen cloned and identified. These accessory molecule ligand genes encodeaccessory molecules which all have the configuration of Type II membraneproteins and exhibit varying degrees of homology with other accessorymolecule ligand genes. For example, the accessory molecule ligand genesencoding both murine CD40 ligand and human CD40 ligand have beenisolated. See, Armitage et al., Nature, 357:80-82 (1992) and Hollenbaughet al., EMBO J., 11:4313-4321 (1992).

CD40 and its ligand, CD40 ligand are critical components of a normalimmune response. CD40 mediated signals induce immune lymphocytes toproliferate and differentiate and become potent antigen presentingcells. Malignant or neoplastic B cells are poor antigen presenter cellsand are unable to stimulate a vigorous allogeneic mixed lymphocytereaction. Successful cross linking of CD40 molecules on immune cellsresults in a strong allogeneic mixed lymphocyte reaction suggesting astrong immune reaction. Various soluble CD40 ligands or antibodiesspecific for CD40 have been used to potentially cross link CD40. Thesesoluble CD40 ligands and CD40-specific antibodies are not optimal forcross linking the CD40 molecules on antigen presenting cells and do notwork as effectively as CD40 ligand expressed on a cell membrane toproduce strong stimulation of antigen presenting cells. These methodsare also difficult to implement because large amounts of CD40 ligandconstructs or antibodies must be isolated which is difficult andtime-consuming work. Other strategies to utilize CD40 ligand in solutionor as a membrane bound molecule including transformation of fibroblastswith CD40 ligand to produce cultured cells which are then used topresent antigen are not amenable to in vivo human clinical protocols.

CD95 (Fas) interaction with its ligand (Fas-ligand, or FasL) functionsto limit the duration of the immune response and/or life-span ofactivated lymphocytes. Apoptosis induced by Fas-FasL binding serves toclear activated self-reactive lymphocytes. Problems caused by alteringthis pathway have been demonstrated in animals with defects inFas<->Fas-ligand interactions. Mice having mutations, which inactivateCD95 or FasL, develop numerous disorders including autoimmune pathologyresembling that seen in patients with rheumatoid arthritis (RA) orsystemic lupus. Zhang, et al., in J. Clin. Invest. 100:1951-1957 (1997)show that injection of FasL-expressing virus, into the joints of micewith collagen-induced-arthritis, results in apoptosis of synovial cellsand relief of arthritis symptoms. Expression of Fas ligand allowsclearance of activated cells which play a role in the pathogenesis ofautoimmune disease. Therefore, a gene therapy strategy for introducingFasL into the joints of rheumatoid arthritis patients could function toimprove disease pathology by leading to destruction of the infiltratingmononuclear cells.

Administration of soluble accessory molecules and accessory moleculeligands has been shown to trigger or to be associated with adversephysiological effects. For example, treatment of mice, having wild-typeCD40-receptor expression, with soluble CD40L-CD8 fusion protein resultedin a pulmonary inflammatory response. This was not observed in mice inwhich the gene for the CD40 receptor had been knocked out. Theseexperiments, described in Wiley, J. A. et al., Journal of Immunology158:2932-2938 (1997), support in vitro data which suggest that CD40ligation can result in inflammatory responses.

Direct administration of purified recombinant soluble Tumor NecrosisFactor (either α or β) results in shock and tissue injury, as. describedin Tracey, K. J., and A. Cerami, Annu. Rev. Med. 45:491-503 (1994).Within minutes after acute intravenous or intra-arterial administrationof TNF, a syndrome of shock, tissue injury, capillary leakage syndrome,hypoxia, pulmonary edema, and multiple organ failure associated with ahigh mortality ensues. Chronic low dose of TNF causes anorexia, weightloss, dehydration and depletion of whole-body protein and lipid.

Soluble Fas ligand and receptor have also been shown to be associatedwith tissue damage and other adverse effects. CD95, the Fas receptor, isa mediator of apoptosis. Fas ligand induces apoptosis by binding to Fasreceptor. As shown in Galle, P. R., et al., J. Exp. Med. 182:1223-1230(1995) administering an agonistic anti-Fas antibody resulted in liverdamage to mice. Mice injected intraperitoneally with the agonisticantibody died within several hours, and analyses revealed that severeliver damage by apoptosis was the most likely cause of death.

The role of soluble Fas ligand (FasL), in the pathogenesis of systemictissue injury in aggressive lymphoma is described in Sato, K. et al.,British Journal of Haematology, 94:379-382 (1996). The findingspresented in this report indicate that soluble FasL is directlyassociated with the pathogenesis of liver injury and pancytopenia.

CD27, the receptor for the accessory molecule ligand, CD70, was shown,in a report written by van Oers, et al., in Blood 82:3430-3436 (1993),to be associated with B cell malignancies.

The above findings all contraindicate the administration of solubleaccessory molecule ligands, highlighting the need for therapies thatincrease the levels of these molecules without resulting in an elevationof their soluble forms.

Despite the wealth of information regarding accessory molecule ligandgenes and their expression on the surface of various immune cells, theexact mechanism by which the accessory molecule ligand genes areregulated on antigen presenting cells is not yet known. Without specificknowledge of the regulation of expression of accessory molecule ligandgenes on these antigen presenting cells, altering the immune response byvarying expression of an accessory molecule ligand gene has to date notbeen possible. Without any specific knowledge as to how to regulate theexpression of an accessory molecule ligand gene on an antigen presentingcell, it is not possible to alter the immune response towards malignantcells. Thus, there was a need for a method of increasing the expressionof an accessory molecule ligand gene on normal and malignant cellsincluding antigen presenting cells.

Further, without the ability to regulate the expression of accessorymolecule ligands, it is not possible to alter the immune clearance ofthese cells.

SUMMARY OF THE INVENTION

The present invention fills these needs by providing novel expressionvectors containing accessory molecule ligand genes and methods forintroducing those genes into normal and malignant antigen presentingcells thereby allowing the alteration of an immune response, thetreatment of autoimmune diseases and the treatment of variousneoplasias. This invention provides vectors, including gene therapyvectors which contain accessory molecule ligand genes. These vectorsalso contain the additional genetic elements, such as promoters,enhancers, polyadenylation signals (3′ ends), which allow that vector tobe successfully placed within the cell and to direct the expression ofthe accessory molecule ligand gene in a cell. Such gene therapy vectorsare capable of transforming animal cells directly and therebyintroducing the accessory molecule ligand gene into the cells of thatanimal in a form which can be utilized to produce accessory moleculeligands within that cell.

In other aspects of the present invention, the function of an accessorymolecule ligand is modified by altering the half life of the molecule onthe cell surface or by changing the level of expression of that moleculeon the cell surface. In preferred embodiments, the present inventionprovides accessory molecule ligands which are modified to improve thestability of such accessory molecule ligands on the cell surface. Suchincreased stability may be accomplished using any of the disclosedmethods of molecules described in this application, including chimericmolecules and molecules into which mutations have been introduced atleast one location. The present invention also contemplates increasingthe expression of such a molecule.

The present invention also provides gene therapy vectors containing theaccessory molecule ligand genes which are chimeric in that portions ofthe gene are derived from two separate accessory molecule ligands whichmay or may not be from different species. The accessory molecule ligandgenes of the present invention include genes which encode molecules ofthe tumor necrosis factor (TNF) family. The molecules which make up theTNF family include TNF_(α), TNF_(β), CD40 ligand, Fas ligand, CD70, CD30ligand, 41BB ligand (4-1BBL), nerve growth factor and TNF-relatedapoptosis inducing ligand (TRAIL). In some embodiments of the presentinvention, the chimeric accessory molecule ligand genes of the presentinvention contain at least a portion of a murine accessory moleculeligand gene together with portions of accessory molecule ligand genesderived from either mouse, humans or other species. Some preferredembodiments of the present invention utilize murine CD40 ligand genesand chimeric CD40 ligand genes containing at least a segment of themurine CD40 ligand gene together with at least a segment of the humanCD40 ligand gene. The present invention contemplates chimeric accessorymolecule ligand genes wherein segments from the accessory moleculeligand gene of one species have been interchanged with segments from asecond accessory molecule ligand gene which may optionally be from adifferent species. For example, in one preferred embodiment, the murineCD40 ligand gene transmembrane and cytoplasmic domains have beenattached to the extracellular domains of human CD40 ligand gene.

The present invention contemplates gene therapy vectors which arecapable of directly infecting the human, mammal, insect, or other cell.The use of such gene therapy vectors greatly simplifies inserting anaccessory molecule ligand gene into those cells. The contemplated genetherapy vectors may be used in vivo or in vitro to infect the desiredcell and are particularly useful for infecting malignant cells to effectsustained high-level expression of a physiologic ligand.

The present invention also contemplates animal, mammal, and human cellscontaining a gene therapy vector which includes an accessory moleculeligand gene and sufficient genetic information to express that accessorymolecule ligand within that cell. In preferred embodiments, the presentinvention also contemplates human neoplastic antigen presenting cellswhich contain the gene therapy vectors of the present invention orcontain an accessory molecule ligand gene together with a promoter and3′ end region.

The present invention also contemplates human cells and human neoplasticcells containing a gene therapy vector which includes a chimericaccessory molecule ligand gene. The present invention also contemplatesbacterial cells or animal cells containing accessory molecule ligandgenes, chimeric accessory molecule ligand genes, murine accessorymolecule ligand genes, human accessory molecule ligand genes, the genetherapy vectors of the present invention, the vectors of the presentinvention, and a chimeric accessory molecule ligand gene together with aheterologous promoter, enhancer or polyadenylation sequence.

The present invention also contemplates methods of altering immuneresponse within a human patient or the immunoreactivity of human cellsin vivo by introducing a gene which encodes an accessory molecule ligandgene into the human cells so that that accessory molecule ligand isexpressed on the surface of those human cells. This method includes theintroduction of the accessory molecule ligand gene as part of a genetherapy vector or in association with a heterologous or native promoter,enhancer or polyadenylation signal. Some preferred embodiments of thepresent invention utilize introduction of Fas ligand genes and chimericFas ligand genes, constructed as contemplated above for CD40, into humancells to alter their immunoreactivity. The present invention alsoincludes methods in which such accessory molecule ligand genes areinserted into cells which have the accessory molecule to which theaccessory molecule ligand binds on the surface of the cell into whichthe accessory molecule ligand gene.

The present methods of altering immunoreactivity are applicable to alltypes of human, animal, and murine cells including human neoplasticcells such as human lymphomas, leukemias and other malignancies. Inpreferred embodiments, this method is used to introduce the geneencoding the accessory molecule ligand into potential antigen presentingcells of a human patient or cell which can stimulate bystanding antigenpresenting cells. Such antigen presenting cells include monocytes,macrophages, B cells, Langerhans cells, interdigitating cells,follicular dendritic cells, Kupffer cells, and the like. The variousantigen presenting cells may be present as part of a known malignancy ina human patient such as leukemias, lymphomas, acute monocytic leukemia(AML), chronic lymphocytic leukemia (CLL), acute myelomonocytic leukemia(AMML), chronic myelogenous or chronic myelomonocytic leukemia (CMML)and thus would include all tumors of any cell capable of presentingantigen to the human or animal immune system or are capable ofstimulating bystanding antigen presenting cells. The present inventionalso contemplates modulating the immune system by introducing genesencoding an accessory molecule ligand gene of the present invention intoany number of different cells found in a patient, including musclecells, skin cells, stromal cells, connective tissue cells, fibroblastsand the like.

The present invention also contemplates methods of treating neoplasiasin either a human patient or an animal patient. In one preferredembodiment, the method comprises isolating the neoplastic cells from thehuman or animal patient and inserting into those isolated cells the genewhich encodes the chimeric accessory molecule ligand or the accessorymolecule ligand so that that molecule is expressed on the cell surfaceof those neoplastic cells or other somatic cells. The neoplastic cellsare then infused back into the human or animal patient and may thenparticipate in an enhanced immune response.

The present invention also contemplates the co-infection orco-introduction of the accessory molecule ligand gene together with agene which encodes a tumor or carcinoma specific antigen. Thiscombination of molecules are then expressed on the surface of theneoplastic cells and when those cells are introduced into the patientlead to the rapid immune response resulting in the destruction of thosecells.

The present methods also include directly introducing the gene therapyvector or other vector carrying the accessory molecule ligand genedirectly into the tumor or tumor bed of a patient. Upon entering thetumor bed of the patient, the gene therapy vector or other vector enterthe cells present in the tumor or tumor bed and then express theaccessory molecule ligand gene on the surface of those cells. Thesecells then are able to participate fully in the human immune or animalimmune response.

The present invention also contemplates methods of augmenting an immuneresponse to a vaccine. The present method of vaccinating an animalagainst a predetermined organism or antigen by administering to thatanimal a vaccine which has a genetic vector containing an accessorymolecule ligand gene. Other embodiments of the present invention includevaccinating an animal by administering two separate genetic vectors, onecontaining the antigens from the organism to which immunity is desiredby isolating the cells of the target animal and contacting with thosecells a vector encoding at least one antigen from a predeterminedorganism so that the antigen is expressed by the cells and alsocontacting those cells with a different vector which expresses theaccessory molecule ligand gene on the surface of the animal's antigenpresenting cells. Together these two separate vectors produce avaccination which is much stronger and of longer duration than isvaccination with antigen alone.

The present methods of vaccination are applicable to vaccinationsdesigned to produce immunity against a virus, a cell, a bacteria, anyprotein or a fungus. The present methods are also applicable toimmunization against various carcinomas and neoplasias. In theseembodiments, the tumor antigen against which immunity is desired isintroduced into the animal together with the genetic vector containingthe accessory molecule ligand gene.

The present invention also contemplates methods of treating arthritisutilizing a gene therapy vector encoding an accessory molecule ligand.Of particular interest for use with arthritis is the Fas ligand moleculein which the expression of Fas ligand activity has been increased in thejoint and/or the stability of the Fas ligand activity on cells withinthe joint enhanced. In other embodiments, the present inventioncontemplated methods of treating arthritis utilizing chimeric accessorymolecule ligands and chimeric accessory molecule ligand genes. Thepresent invention also contemplates both ex vivo therapy and in vivotherapy of arthritis utilizing the expression vectors of the presentinvention together with the Fas ligand and modified versions of thatmolecule including chimeric molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 is a diagram showing a number of accessory moleculeligand genes and Domains I-IV of those genes as deduced from sequencedata.

FIG. 2. FIG. 2 is a diagram showing example chimeric accessory moleculeligand genes. The domains derived from the murine accessory module areshown shaded.

FIG. 3. FIG. 3 shows the amount of either mouse or human CD40 ligandfound on the surface of Hela or CLL cells infected with gene therapyvectors containing the genes encoding these molecules. FIG. 3A showsuninfected Hela cells (shaded) and Hela cells infected with a genetherapy vector encoding murine CD40 ligand. FIG. 3B shows uninfectedHela cells (shaded) and Hela cells infected with a gene therapy vectorencoding human CD40 ligand. FIG. 3C shows uninfected CLL cells (shaded)and CLL cells infected with a gene therapy vector encoding murine CD40ligand. FIG. 3D shows uninfected CLL cells (shaded) and CLL cellsinfected with a gene therapy vector encoding human CD40 ligand.

FIG. 4. FIG. 4 shows histograms of the increased expression of CD54(FIG. 4B) and CD80 (FIG. 4D) on CLL cells into which a gene therapyvector containing the accessory molecule ligand gene (murine CD40 ligandgene) has been introduced. The shaded graph indicates control stain inFACS analysis and the open graph indicates staining with monoclonalantibodies immunospecific for either CD54 (FIGS. 4A and 4B) or CD80(FIGS. 4C and 4D).

FIG. 5. FIG. 5 shows the cell proliferation as measured by ³H-TdRincorporation of allogeneic T cells in response to various stimulationregimes. The CLL cells containing a gene therapy vector expressing anaccessory molecule ligand gene (the murine CD40 ligand gene) wereintroduced, stimulating allogeneic T cells to proliferate.

FIG. 6. FIG. 6 shows the production of gamma interferon (IFN_(γ)) byallogeneic T cells stimulated with CLL cells containing an accessorymolecule ligand gene.

FIG. 7. FIG. 7 shows the treatment of a neoplasia in an animal using agene therapy vector containing an accessory molecule ligand gene of thepresent invention. The open squares show mice immunized with neoplasticcell not expressing an accessory molecule ligand of the presentinvention. Mice immunized with neoplastic cells expressing an accessorymolecule ligand of the present invention are shown as the horizontalline at the top of the Figure and show no morbidity.

FIG. 8. FIG. 8 shows the production levels and stabilities of CD40ligand and CD40 ligand transcript in CLL (upper graph) and normal bloodmononuclear cells (lower graph).

FIG. 9. FIG. 9 shows the time course of transgene expression in CLL Bcells infected with the accessory molecule ligand (CD40 ligand). TheMFIR (mean fluorescence intensity ratio), comparing the fluorescenceintensity of CD19⁺ CLL cells stained with PE-labeled CD40 ligand versusthe same stained with a PE-labeled isotype control mAb at each timepoint, are represented by the closed circles connected by solid linesaccording to the scale provided on the left-hand ordinate.

FIG. 10. FIG. 10 shows changes in surface antigen phenotype of CLL Bcells infected with a gene therapy vector containing an accessorymolecule ligand, CD40 ligand. Shaded histograms represent staining ofuninfected CLL cells (thin lines) stained with nonspecific controlantibody, open histograms drawn with thin lines represent uninfected CLLcells stained with FITC-conjugated specific mAb, and open histogramsdrawn with thick lines (labeled CD154-CLL) represent CLL cells infectedwith the accessory molecule ligand gene therapy vector and stained withFITC-conjugated specific mAb.

FIG. 11. FIG. 11 shows levels of CD27 produced in CLL cells infectedwith a gene therapy vector containing an accessory molecule ligand. FIG.11A shows that CD40L-infected CLL (CD154-CLL) cells express reducedlevels of surface CD27. Open histograms represent staining ofnon-infected CLL cells (thin lines) or infected CLL (thick lines) withFITC-conjugated αCD27 mAb, respectively. FIG. 11B shows production ofsoluble form of CD27 by CLL B cells.

FIG. 12. FIG. 12 shows allogeneic T cell responses induced by CLL cellsinfected with a gene therapy vector containing an accessory moleculeligand (CD40 ligand, also called CD154). FIG. 12A indicates theconcentration of IFN_(γ) in the supernatants after stimulation ofallogeneic T cells with CLL cells containing the accessory moleculeligand. FIG. 12B shows cell proliferation, as assessed by incorporationof ³H-thymidine. FIGS. 12C and 12D show secondary allogeneic T cellresponses induced by CLL containing the accessory molecule ligand.

FIG. 13. FIG. 13 depicts autologous T cell responses induced by CLL Bcells containing the accessory molecule ligand, CD40 ligand or CD154,and controls. FIG. 13A shows incorporation of ³H-thymidine by autologousT cells co-cultured with the CLL cells. FIG. 13B shows the levels ofhuman IFN_(γ) produced by autologous T cells co-cultured with the CLLcells. In FIG. 13C, the CTL activities of autologous T cells induced byCLL B cells containing the accessory molecule ligand are graphed.

FIG. 14. FIG. 14 shows specificity of CTL for autologous CLL B cells.IFN_(γ) concentration was assessed in the supernatants after 48 h ofculture (FIG. 14A), and cytolytic activity was assessed at 3 h ofculture (FIG. 14B). In FIG. 14C, mAb were added to the autologousleukemia target cells prior to the CTL assay.

FIG. 15. FIG. 15 shows that intercellular stimulation plays a role inproduction of the phenotypic changes observed in CLL cells expressingthe accessory molecule ligand. In FIG. 15A, the effect of culturedensity on the induced expression of CD54 and CD80 following infectionwith a gene therapy vector containing the accessory molecule ligand(CD40 ligand, CD154) is shown. Shaded histograms represent staining ofleukemia B cells with a FITC-conjugated isotype control mAb. Openhistograms represent CD154-CLL B cells, cultured at high or low density(indicated by arrows), and stained with a FITC-conjugated mAb specificfor CD54 or CD80. FIG. 15B shows inhibition of CD154-CLL cell activationby anti-CD154 mAb. FIGS. 15C and 15D depict expression of immuneaccessory molecules on bystander non-infected CLL B cells induced by CLLcells expressing the accessory molecule ligand. Shaded histogramsrepresent staining with PE-conjugated isotype control mAb.

FIG. 16. FIG. 16 shows that the vector encoding an accessory moleculeligand enhances immunization against β-gal in mice. FIG. 16A shows thatmice that received intramuscular injections of the pCD40L vectorproduced significantly more antibodies to β-gal than did mice injectedwith either the non-modified pcDNA3 vector or pCD40L. FIG. 16B, ELISAanalyses of serial dilutions of sera collected at d28, shows that miceco-injected with placZ and pCD40L had an eight-fold higher mean titer ofanti-β-gal antibodies at d28 than mice treated with placZ+pcDNA3.

FIG. 17. FIG. 17 shows analysis of the IgG₁ and IgG_(2a) immuneresponses to intramuscular plasmid DNA immunizations with and without avector, pCD40L, encoding an accessory molecule ligand. IgG_(2a)anti-β-gal antibodies predominated over IgG₁ subclass antibodies in thesera of mice injected with either placZ and pcDNA3 or placZ and pCD40L.In contrast, BALB/c mice injected with β-gal protein developedpredominantly IgG₁ anti-β-gal antibodies, and no detectable IgG_(2a)anti-β-gal antibodies.

FIG. 18. FIG. 18 shows the comparison between injection of mice with avector, pCD40L, encoding an accessory molecule ligand, at the same anddifferent sites as placZ. Adjuvant effect of pCD40L requiresco-injection with placZ at the same site.

FIG. 19. FIG. 19 shows that co-injection into dermis of a vectorencoding an accessory molecule ligand, pCD40L, with placZ enhances theIgG anti-β-gal response in BALB/c mice.

FIG. 20. FIG. 20 shows that a vector encoding an accessory moleculeligand, pCD40L, enhances the ability of placZ to induce CTL specific forsyngeneic β-gal-expressing target cells. Splenocyte effector cells,taken from mice which had received injections of placZ and pCD40L,specifically lysed significantly more cells than did splenocytes frommice that received control injections.

FIG. 21. FIG. 21 shows downmodulation of human CD40L, but not murineCD40L, in lung tumor cell lines that express CD40.

FIG. 22. FIG. 22A shows that CD40 binding induces enhanced expression ofthe tumor cell surface markers CD95 (Fas), CD54 (ICAM-1), and MHC-I, inlung tumor cell lines. FIG. 22B shows downmodulation of human CD40L byCD40-positive tumor cells.

FIG. 23. FIG. 23 shows the inhibition of Fas ligand expression bylymphocytes in the presence of RA synovial fluid.

FIG. 24. FIG. 24 shows an outline for a clinical trial of an accessorymolecule ligand (CD40L) gene therapy treatment for B cell CLL.

FIG. 25. FIG. 25 shows a sequence line-up of human Fas ligand with humanFas ligand in which Domain III is replaced by Domain III of murine Fasligand. The top protein sequence is native human Fas ligand. Domain IIIis underlined with the dotted line. The double underline indicates aputative MMP cleavage site. The bottom protein sequence is that ofchimeric human-mouse Fas ligand. Domain III of the mouse Fas ligand(underlined with dotted line) is substituted for Domain III of human Fasligand. The numbers correspond to the amino acid sequence number using 1for the start of the polypeptide sequence. The number of the firstnucleotide base for the codon encoding the amino acid is 1+3x(n−1),where n is the amino acid sequence number.

FIG. 26. FIG. 26 shows a sequence line-up of human Fas ligand with humanFas ligand in which Domain III has been replaced with Domain III ofhuman CD70. The top protein sequence is native human Fas ligand, and thebottom sequence is that of chimeric Fas ligand, in which Domain III ofhuman CD70 has been substituted for Fas Domain III. Other markings areused similarly as in FIG. 25.

FIG. 27. FIG. 27 shows a sequence line-up of human Fas ligand with humanFas ligand in which Domain I has been replaced with Domain III of humanCD70. The top protein is native human Fas ligand, and the bottom animal.Preferred types of malignant or neoplastic cells include any malignantantigen-presenting cell. In some preferred embodiments, these malignantantigen presenting cells have at least low levels of CD40 present on thecell surface.

As used herein, the term “neoplastic human cells” is defined to meanhuman cells which are neoplastic including but not limited to antigenpresenting cells, any neoplastic cell which may function as an antigenpresenting cell or function to facilitate antigen presentation,neoplastic monocytes, neoplastic macrophages, neoplastic B cells,neoplastic dendritic cells, neoplastic Langerhans cells, neoplasticinterdigitating cells, neoplastic follicular dendritic cells, orneoplastic Kupffer cells and the like. The definition of neoplastichuman cells includes those cells which are associated with neoplasticcells in the tumor bed of human patients.. Typically, the neoplastichuman cells are either leukemias, lymphomas, AML, ALL, AMML, CML, CMML,CLL other tumors of antigen presenting cells or breast, ovarian or lungneoplastic cells. It is also contemplated that the accessory moleculeligand genes or chimeric accessory molecule ligand genes of the presentinvention may be inserted into somatic cells. These somatic cells can becreated by a genetic engineering process which has introduced into thosecells genes which encode molecules which render those cells capable ofpresenting antigen to the immune-system.

As used herein, the term “chimeric gene” is defined to mean a gene inwhich part of the gene is derived from a second different gene andcombined with the first gene so that at least a portion of each gene ispresent in the resulting chimeric gene. A gene may be chimeric if anyportion of the sequence which encodes the resulting protein sequence isthat of chimeric Fas ligand, in which Domain III has been replaced withDomain I of human CD70. Other markings are used similarly as in FIG. 25.

FIG. 28. FIG. 28 shows the amino acids around and at known matrixmetalloproteinase (MMP) cleavage sites, as described in Smith, M. M. etal., Journal of Biol. Chem. 270:6440-6449 (95) and Nagase, H., and G. B.Fields, Biopolymers (Peptide Science) 40:399-416 (96). The cleavage siteis indicated with an arrow.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated in their entirety byreference.

I. Definitions

An “accessory molecule ligand gene” is a gene which encodes all or partof an accessory molecule ligand. The gene comprises at least thenucleotide sequence required to encode the functional portion of anaccessory molecule ligand. The gene may optionally include such geneticelements as promoters, enhancers and 3′ ends. The accessory moleculeligand gene is derived from a ligand which is a member of the tumornecrosis factor (TNF) family, including CD40 ligand, Fas ligand, CD70,TNF_(α), TNF_(β), CD30 ligand, 4-1BB ligand (4-1BBL), nerve growthfactor and TNF-related apoptosis inducing ligand (TRAIL). As usedherein, the term “accessory molecule ligand gene” includes chimericaccessory molecule ligand genes as defined below.

As used herein, the term “malignant cells or neoplastic cells,” isdefined to mean malignant or cancerous cells which are found in a humanpatient or an protein is derived from a second and different gene.Typical chimeric genes include genes in which specific functionaldomains from one gene have been transferred to a second gene and replacethe analogous domains of that second gene. For example, the resultingchimeric gene may have one domain derived from a murine gene and severaldomains derived from a human gene. These domains may range in size from5 amino acids to several hundred amino acids. Other examples of chimericaccessory molecule ligand genes include genes which contain nucleotidesencoding amino acids not found in any naturally occurring accessorymolecule ligand gene. Examples of chimeric genes and potential variouscombinations of domains are numerous and one of skill in the art willunderstand that no limit is placed on the amount of one gene that mustbe present in a second gene to render it chimeric.

As used herein, the term “murine CD40 ligand gene” is defined to mean anaccessory molecule ligand gene which is derived from a murine CD40ligand gene. Examples of such murine CD40 ligand genes include the geneisolated by Armitage et al., Nature, 357:80-82 (1992) and other genesderived from murine origin which hybridize to the gene described byArmitage et al. under low stringency hybridization conditions.

As used herein, the term “vector or genetic vector” is defined to mean anucleic acid which is capable of replicating itself within an organismsuch as a bacterium or animal cell. Typical genetic vectors include theplasmids commonly used in recombinant DNA technology and various virusescapable of replicating within bacterial or animal cells. Preferred typesof genetic vectors includes plasmids, phages, viruses, retroviruses, andthe like.

As used herein, the term “gene therapy vector” is defined to mean agenetic vector which is capable of directly infecting cells within ananimal, such as a human patient. A number of gene therapy vectors havebeen described in the literature, and include, the gene therapy vectordescribed in Cantwell et al., Blood, In Press (1996) entitled“Adenovirus Vector Infection of Chronic Lymphocytic Leukemia B Cells.”Such vectors have been described for example by Woll, P. J. and I. R.Hart, Ann. Oncol., 6 Suppl 1:73 (1995); Smith, K. T., A. J. Shepherd, J.E. Boyd, and G. M. Lees, Gene Ther., 3:190 (1996); Cooper, M. J., Semin.Oncol., 23:172 (1996); Shaughnessy, E., D. Lu, S. Chatterjee, and K. K.Wong, Semin. Oncol., 23:159 (1996); Glorioso, J. C., N. A. DeLuca, andD. J. Fink, Annu. Rev. Microbiol., 49:675 (1995); Flotte, T. R. and B.J. Carter, Gene Ther., 2:357 (1995); Randrianarison-Jewtoukoff, V. andM. Perricaudet, Biologicals., 23:145 (1995); Kohn, D. B., Curr. Opin.Pediatr., 7:56 (1995); Vile, R. G. and S. J. Russell, Br. Med. Bull.,51:12 (1995); Russell, S. J., Semin. Cancer Biol., 5:437 (1994); andAli, M., N. R. Lemoine, and C. J. Ring, Gene Ther., 1:367 (1994). Allreferences cited herein are hereby incorporated by reference.

II. Genetic Vectors and Constructs Containing an Accessory MoleculeLigand Gene

A. Accessory Molecule Ligand Genes

In one embodiment of the present invention, preferred gene therapyvectors contain an accessory molecule ligand gene. This accessorymolecule ligand gene may be derived from any source and may includemolecules which are man-made and do not appear in nature. The presentinvention contemplates accessory molecule ligand genes which are derivedfrom the genes encoding molecules within the tumor necrosis family (TNF)which includes the genes encoding: murine CD40 ligand, human CD40ligand, Fas ligand, TNF_(α), TNF_(β), CD30 ligand, 4-1BB ligand, nervegrowth factor, CD70, TNF-related apoptosis inducing ligand (TRAIL) andchimeric accessory molecule ligands. The nucleotide sequence of oneaccessory molecule ligand, the sequence of at least one form of themurine CD40 ligand gene, has been determined and is listed as SEQ IDNO: 1. The present invention contemplates the use of any accessorymolecule ligand gene which is homologous to the sequence present in SEQID NO: 1, and thus hybridizes to this sequence at low stringencyhybridization conditions. One of skill in the art will understand thataccessory molecule ligand genes, including murine CD40 ligand gene,useful in the present invention may be isolated from various differentmurine strains.

The nucleotide sequence of a human CD40 ligand gene has been determinedand is shown as SEQ ID NO: 2. The present invention contemplates the useof any accessory molecule ligand gene which is homologous to SEQ ID NO:2, and thus hybridizes to this sequence at low stringency conditions.One of ordinary skill in the art will understand that the accessorymolecule ligand genes, including the human CD40 ligand genes, useful inthe present invention, may vary depending on the individual from whichthe gene is isolated and such variations may prove useful in producingunique accessory molecule ligand genes. The present inventioncontemplates the use of the domains, sub-domains, amino acid ornucleotide sequence of the human CD40 ligand and/or human CD40 ligandgene as part of a chimeric accessory molecule ligand or chimericaccessory molecule ligand gene.

The nucleotide sequence of a bovine CD40 ligand gene has been determinedand is shown as SEQ ID NO: 8. The present invention contemplates the useof any accessory molecule ligand gene which is homologous to SEQ ID NO:8, and thus hybridizes to the sequence at low stringency conditions. Oneof ordinary skill in the art will understand that the accessory moleculeligand genes, including the bovine CD40 ligand genes, may vary dependingon the individual animal from which the gene is isolated and that suchvariations may prove useful in producing unique accessory moleculeligand genes.

The nucleotide sequence of human TNF_(α) and human TNF_(β) have beendetermined and are shown as SEQ ID NOS: 9 and 10, respectively. Thepresent invention contemplates the use of any accessory molecule ligandgene which is homologous to either human TNF_(α) or human TNF_(β) (SEQID NOS: 9 and 10, respectively), and thus hybridizes to these sequencesat low stringency conditions. The accessory molecule ligand genes usefulin the present invention, including the human TNF_(α) and TNF_(β) genes,may vary depending on the particular individual from which the gene hasbeen isolated and these variations may prove useful in producing uniqueaccessory molecule genes.

The nucleotide sequence of porcine TNF_(α) and TNF_(β) have beendetermined and are shown as SEQ ID NO: 11. The present inventioncontemplates the use of any accessory molecule ligand gene which ishomologous to either SEQ ID NO: 11, and thus would hybridize to thesesequences at low stringency conditions. One of ordinary skill in the artwill understand that the accessory molecule ligand genes, including theporcine TNF_(α) and TNF_(β) genes, may vary depending on the particularanimal from which the gene is isolated and that such variation may proveuseful in producing unique accessory molecule genes.

The nucleotide sequence of a murine TNF_(α) gene has been determined andis shown as SEQ ID NO: 12. The present invention contemplates the use ofany accessory molecule ligand gene which is homologous to SEQ ID NO: 12,and thus hybridizes to the sequence at low stringency conditions. One ofordinary skill in the art will understand that the accessory moleculeligand genes, including the murine TNF_(α) gene may vary depending onthe individual from which the gene is isolated and that these variationsmay prove useful in producing unique accessory molecule genes.

The nucleotide sequence of human Fas ligand and murine (C57BL/6) Fasligand have been determined and are shown as SEQ ID NOS: 13 and 14,respectively. The nucleotide sequence of murine Balb/c Fas ligand isshown as SEQ ID NO: 31. The present invention contemplates the use ofany accessory molecule ligand gene which is homologous to any of SEQ IDNOS: 13, 14, and 31, and thus hybridizes to the sequences at lowstringency conditions. One of ordinary skill in the art will understandthat the accessory molecule ligand genes, including the human Fas ligandor murine Fas ligand genes may vary depending on the particularindividual or animal from which the gene is isolated and that suchvariations may prove useful in producing any accessory molecule genes.

The nucleotide sequence of a human CD70 gene has been determined and isshown as SEQ ID NO: 15. The murine CD70 gene sequence has also beendetermined, and is shown as SEQ ID NO: 36 and was described by Tesselaaret. al, J. Immunol. 159:4959-65(1997). The present inventioncontemplates the use of any accessory molecule ligand gene which ishomologous to SEQ ID NO: 15 or 36, and thus hybridizes to this sequenceat low stringency conditions. One of ordinary skill in the art willunderstand that the accessory molecule ligand genes, including the humanCD70 gene may vary depending on the individual from which the gene isisolated and that these variations may prove useful in producing uniqueaccessory molecule ligand genes.

The nucleotide sequence of human CD30 ligand gene has been determinedand is shown as SEQ ID NO: 16. The present invention contemplates theuse of any accessory molecule ligand gene which is homologous to SEQ IDNO: 16, and thus hybridizes to this sequence at low stringencyconditions. One of ordinary skill in the art will understand that theaccessory molecule ligand genes, including the human CD30 ligand gene,may vary depending on the individual from which the gene is isolated andthat such variations may prove useful in producing unique accessorymolecule ligand genes.

The present invention also contemplates variations and variants of thenucleotide sequences of the accessory molecule ligand genes providedherein which are caused by alternative splicing of the messenger RNA.This alternative splicing of the messenger RNA inserts additionalnucleotide sequences which may encode one or more optional amino acidsegments which in turn allows the accessory molecule ligand encoded tohave additional properties or functions.

The nucleotide sequence of a human and mouse 4-1BBL have been determinedand are shown as SEQ ID NOS: 17 and 18, respectively. The presentinvention contemplates the use of any accessory molecule ligand genewhich is homologous to either SEQ ID NOS: 17 or 18, and thus hybridizesto these sequences at low stringency conditions. One of ordinary skillin the art will understand that accessory molecule ligand genes,including the human 4-1BBL gene may vary depending on the individualfrom which it is isolated and that such variations may prove useful inproducing unique accessory molecule ligand genes.

The present invention also contemplates chimeric accessory moleculescontaining any domain, sub-domain portion, or amino acid sequenceencoded by the following genes: bovine TNF-α (SEQ ID NO: 21), murineCD40 ligand (SEQ ID NO: 22), human nerve growth factor-β (SEQ ID NO:23), murine nerve growth factor (SEQ ID NO: 24), rat Fas ligand (SEQ IDNO: 25), human TNF-related apoptosis inducing ligand (TRAIL) (SEQ ID NO:41, Genbank accession number U37518), murine TNF-related apoptosisinducing ligand (TRAIL) (SEQ ID NO: 42, Genbank accession numberU37522), murine CD30-Ligand (SEQ ID NO: 43), human 4-1BBL (SEQ ID NO:17), and murine 4-1BBL (SEQ ID NOS: 44 and 18). The present inventionalso contemplates chimeric accessory molecules which utilize genesencoding amino acid sequences homologous to these sequences.

The present invention contemplates chimeric accessory molecule ligandgenes which are comprised of a nucleotide segment derived from oneaccessory molecule ligand gene operatively linked to a nucleotidesequence derived from a different accessory molecule ligand gene orother gene.

For example, chimeric accessory molecule ligand genes are contemplatedwhich are comprised of a segment of the murine CD40 ligand gene whichhas been operatively linked to at least one other additional genesegment derived from a different accessory molecule ligand gene. Thesize of the particular segment derived from the different accessorymolecule ligand gene may vary from a nucleotide sequence encoding a fewamino acids, a sub-domain of the accessory molecule ligand, a domain ofthe accessory molecule ligand or more than a domain of an accessorymolecule ligand. Other chimeric accessory molecules of the presentinvention are comprised of an accessory molecule ligand gene into whichnucleotides encoding an amino acid segment which is not found as part ofa naturally occurring accessory molecule ligand have been inserted. Thisamino acid segment may be artificially created or derived from a proteinfound in nature. The chimeric accessory molecule ligand gene encodes achimeric amino acid sequence and thus a chimeric accessory moleculeligand encoded may possess unique properties in addition to theproperties found on the individual segments derived from the differentaccessory molecule ligand genes. The chimeric accessory molecule ligandgene may encode an accessory molecule ligand which has propertiesderived from the accessory molecule ligand used to construct thechimeric gene.

Each of the accessory molecule ligand genes which are a member of thetumor necrosis factor family have a similar secondary structureconsisting of a number of domains. This domain structure includes afirst domain which is encoded by the 5′ region of the accessory moleculeligand gene. The second domain (Domain II) is the domain which containsthe amino acids which span the cell membrane and is thus called thetransmembrane domain. The third domain (Domain III) is the proximalextracellular domain and these amino acids are the amino acids which arefound proximal to the cellular membrane. The fourth domain (Domain IV),is encoded by the 3′ end of the accessory molecule ligand gene and hasbeen called the distal extracellular domain. The distal extracellulardomain (Domain IV) generally makes up the soluble form of the tumornecrosis factor family molecule. Based on the x-ray crystal structure ofhuman TNF, the predicted secondary structure of the accessory molecule,CD40 ligand has been deduced together with the domain structure of thesemolecules by M. Peitsch and C. Jongeneel, International Immunology,5:233-238 (1993). The secondary structures of the other members of thetumor necrosis factor family were deduced using computer analysistogether with comparison to the human TNF and CD40 ligand domainstructure. In Table I, the domain boundaries of a number of accessorymolecule ligand genes is shown. A diagram of these domains for a numberof these accessory cell molecule ligands is shown in FIG. 1. Theassignments of the domain boundaries are approximate and one of ordinaryskill in the art will understand that these boundaries may vary and yetstill provide useful identification of domains. TABLE I DOMAIN STRUCTUREOF TUMOR NECROSIS FACTOR FAMILY MOLECULES* Domain III Domain IV Domain IDomain II (Proximal (Distal (Cyto- (Trans- Extra- Extra- plasmic)membrane) cellular) cellular) Human CD40 1-42 43-135 136-330 331-786Ligand Murine CD40 1-42 43-135 136-327 328-783 Ligand Bovine CD40 1-4243-135 136-330 331-786 Ligand Human TNF-α 1-87 88-168 169-228 229-699Murine TNF-α 1-87 88-168 169-237 238-705 Porcine TNF-α 1-87 88-168169-228 229-696 Human TNF-β 1-39 40-129 130-153 154-615 Porcine TNF-β1-39 40-126 127-150 151-612 Human Fas  1-237 238-315  316-390 391-843Ligand Murine Fas  1-237 238-309  310-384 385-837 Ligand Human CD70 1-6162-117 118-132 133-579 Murine CD70 1-73 74-123 124-138 139-585 HumanCD30  1-117 118-186  187-240 241-702 Ligand Murine CD30  1-135 136-201 202-255 256-717 Ligand Human 4-1BBL 1-69 70-174 175-210 211-762 Murine4-1BBL  1-237 238-333  334-369 370-927 Human TRAIL 1-39 40-117 118-375376-843 Murine TRAIL 1-51 52-111 112-387 388-873*The Domains above are identified by the nucleotide boundaries of eachdomain using the first nucleotide of the initial methionine of the cDNAas nucleotide number 1.

One of ordinary skill in the art will understand that typical chimericaccessory molecule genes would include genes produced by exchangingdomains or sub-domain segments between, for example, a mouse CD40 ligandgene and a human CD40 ligand gene. For example, chimeric accessorymolecule gene may be constructed by operatively linking Domain I of thehuman CD40 ligand gene to Domains II-IV of the murine CD40 ligand gene.One of ordinary skill in the art will understand the variety of chimericaccessory molecule ligand genes which may be produced using theaccessory molecules identified in Table I. The present invention alsocontemplates chimeric accessory molecules which are not shown in Table Ibut which are shown to have a similar domain structure. Other chimericgenes are also contemplated in which smaller segments (sub-domainsegments) are exchanged between, for example, a murine CD40 ligand geneand a human CD40 ligand gene or a second murine CD40 ligand gene. One ofskill in the art will understand that genes encoding accessory moleculeswill have at least gene segments which correspond to various functionalsegments of an accessory molecule ligand such as the murine CD40 ligandencoded by the murine CD40 ligand gene (SEQ ID NO: 1). It will also beapparent to one of skill in the art that the nucleotide boundariesidentified in Table I may vary considerably from those identified forthe murine CD40 ligand gene (SEQ ID NO: 1) and still define domainswhich are useful in the present invention.

In one preferred embodiment, the chimeric accessory molecule ligand geneis comprised of the nucleotides encoding extracellular domains (DomainsIII and IV) of human CD40 ligand operatively linked to the nucleotidesencoding transmembrane (Domain II) and the nucleotides encodingcytoplasmic domain (Domain I) of the murine CD40 ligand gene. Examplesof such preferred chimeric accessory molecules are shown in FIG. 2. Anexemplary nucleotide sequence for such a gene is SEQ ID NO: 7. In otherchimeric accessory molecule ligand genes of the present invention, thenucleotides encoding the extracellular domains (Domains III and IV) ofthe murine CD40 ligand gene may be operatively linked to nucleotidesencoding the transmembrane (Domain II) and cytoplasmic domain (Domain I)of the human CD40 ligand gene. An exemplary nucleotide sequence for sucha gene is SEQ ID NO: 3. In other preferred chimeric accessory moleculeligand genes of the present invention, the nucleotides encoding theextracellular domains (Domains III and IV) and transmembrane domain(Domain II) of human CD40 ligand are coupled to the nucleotides encodingcytoplasmic domain (Domain I) of murine CD40 ligand gene. An exemplarynucleotide sequence for such a gene is SEQ ID NO: 6. Other chimericaccessory molecule genes contemplated by the present invention comprisethe nucleotides encoding the extracellular domains (Domains III and IV)and transmembrane domain (Domain I) of the murine CD40 ligand geneoperatively linked to the nucleotides encoding cytoplasmic domain of thehuman CD40 ligand gene. An exemplary nucleotide sequence for such a geneis SEQ ID NO: 5. Other chimeric accessory molecule ligand genes arecontemplated by the present invention in which the human CD40 ligandgene extracellular domains (Domain III and IV) is operatively linked tothe murine CD40 ligand gene transmembrane domain (Domain I) which isoperatively linked to the human CD40 ligand gene cytoplasmic domain(Domain I). An exemplary nucleotide sequence for such a gene is SEQ IDNO: 4.

One of ordinary skill in the art will understand that many morecombinations which utilize domains or other selected segments of any ofthe accessory molecule ligand genes including the human CD40 ligandgenes and the mouse CD40 ligand genes are possible. Such additionalchimeric accessory molecule genes would include the following genes:chimeric accessory molecule genes in which the nucleotides encodingDomain I are selected from a particular accessory molecule ligand geneand operatively linked, either directly or by an additional nucleotidesequence to the nucleotides encoding Domain II from a particularaccessory molecule ligand gene. These domains then would be operativelylinked either directly or by an additional nucleotide sequence to thenucleotides encoding Domain III from a particular accessory moleculeligand gene. This molecule would then be operatively linked eitherdirectly or by an additional nucleotide sequence to the nucleotidesencoding Domain IV of a particular accessory molecule ligand gene. Thechimeric accessory molecule ligand gene constructed in this manner mayhave additional nucleotides on either end or between domains which areuseful to provide different amino acids in these positions. One ofordinary skill in the art will understand that these particularcombinations are merely illustrations and that numerous othercombinations could be contemplated in which gene segments comprisingnucleotides encoding less than the entire domain of an accessorymolecule are exchanged between different accessory molecules.

The present invention also contemplates chimeric accessory moleculeligand genes which are comprised of gene segments of mouse or human CD40ligand in combination with gene segments derived from Fas ligand,TNF_(α), TNF_(β), CD70, CD30L, 4-1BBL, nerve growth factor orTNF-related apoptosis inducing ligand (TRAIL). Particularly usefulchimeric accessory molecule ligand genes comprise at least one genesegment which is derived from a murine CD40 ligand gene together withgene segments or a gene segments derived from a different accessorymolecule ligand gene.

The present invention also contemplates chimeric accessory moleculeligand genes in which the accessory molecules produced have beenmodified to remove amino acids within the chimeric accessory moleculethat are used by post-translational mechanisms to regulate the level ofexpression of the accessory molecule or accessory molecule protein on aparticular cell. The sites removed from the chimeric accessory moleculesor chimeric molecule may include amino acids or sites which make upprotease cleavage sites including metallothionine proteases, serineproteases and other proteases that recognize an amino acid sequenceeither specifically or nonspecifically. In particular preferredembodiments, amino acids in Domain III which make up potential or actualrecognition site(s) used by post-translational regulatory mechanismshave been modified or removed.

The present invention also contemplates chimeric accessory moleculeligand genes in which the domains, subdomain fragments or other aminoacid residues have been taken from one accessory molecule ligand geneand moved into a second accessory molecule ligand gene from the samespecies. For example, in this particular embodiment, the human Domain I,and the human Domain II from the CD40 ligand molecule may be operativelylinked to the nucleotides encoding the human Domain III from, forexample, the CD70 molecule which is in turn operatively linked to humanDomain IV for the CD40 ligand molecule. This chimeric accessory moleculetherefore contains human CD40L Domains I, II and IV and human CD70Domain III. An exemplary nucleotide sequence for such a gene is SEQ IDNO: 19. One of ordinary skill in the art will understand that a numberof such combinations using domains from the same species from differentaccessory molecule ligand genes may create a number of chimericaccessory molecule genes which may all have specific activities andproperties.

The present invention contemplates chimeric accessory molecule ligandgenes in which the Domain III of a particular accessory molecule ligandgene has been replaced with a Domain III from a different accessorymolecule ligand gene. In one particularly preferred embodiment, themouse Domain III has been used to replace the human Domain III in theCD40 ligand molecule. This chimeric accessory molecule thereforecontains the human CD40L Domain I, the human CD40L Domain II, mouseCD40L Domain III, and human CD40L Domain IV. An exemplary nucleotidesequence for such a gene is SEQ ID NO: 20.

The present invention also contemplates the use of chimeric accessorymolecules that contain man-made amino acid sequences inserted into or inplace of a portion of a domain or other amino acid sequence of anaccessory molecule gene. These man-made amino acid segments may becreated by selecting any amino acid sequence that may be used to givethe accessory molecule a particular function or to remove anotherundesired function. These man-made amino acid segments are produced byinserting into the accessory molecule ligand gene or chimeric accessorymolecule ligand gene the nucleotide sequences required to encode thoseparticular man-made amino acid segments in the desired positions.Further, the chimeric accessory molecule ligand genes may containnucleotide segments which comprise sub-domain segments of othermolecules or small segments in which amino acids have been changed for adesired purpose. The use of sub-domain nucleotide segments allows theintroduction of short amino acid sequences derived from other moleculesinto chimeric accessory molecules of the present invention. Theincorporation of such short sub-domain segments or amino acid changesinto the accessory molecule ligand allows the introduction of desired orthe removal of undesired features of that molecule.

The identification of domain structures within accessory cell moleculesis well known in the art and generally requires the identification ofcysteine residues within the accessory molecules and the subsequentmapping of disulfide bonds between various cysteine residues. Themapping of various sub-domain segments of an accessory molecule is wellknown in the art and involves analysis of the amino acid sequence of theaccessory molecules and generally involves a comparison of the crystalstructure of tissue necrosis factor with the use of predictivealgorithms thereby producing a predicted structure of a chimericaccessory molecule or an accessory molecule. This predicted structure ofthese molecules can then be used to select various sub-domain portionsof the molecule to be used to construct further chimeric accessorymolecules. Examples of such mapping studies include the studies by M.Pitsch and C. V. Jongeneel, International Immunology, 5:233-238 (1993)and the analysis shown in FIG. 1.

The present invention also contemplates accessory molecule ligand genesand chimeric accessory molecule ligand genes which are truncated andencode less than the full length of the amino acid sequence found in thenative accessory molecule ligand. These truncations may alter theproperties of the accessory molecule ligand gene but some identifiedactivity is maintained. Such truncations may be made by removing a genesegment or gene segments from the accessory molecule gene and typicallywould be performed by removing nucleotides encoding domains which arenot directly involved in the binding of the accessory molecule ligandwith its accessory molecule. These truncated accessory molecule ligandgenes or chimeric truncated accessory molecule ligand genes may containfurther gene segments which encode amino acid segments or domains whichreplace the domains removed from that truncated accessory molecule gene.However, such replacement of the portions of the accessory moleculeremoved by truncation is not necessary.

The chimeric accessory molecule genes of the present invention may beconstructed using standard genetic engineering methods to operativelylink a particular nucleotide sequence from one accessory molecule ligandgene to a different nucleotide sequence derived from the same ordifferent accessory molecule ligand gene. In addition, standard geneticengineering methods may be used to insert man-made nucleotide sequencesor sub-domain nucleotide sequences into the chimeric accessory moleculeligand gene. One of ordinary skill in the art will understand thatvarious methods may be utilized to produce such chimeric accessorymolecule genes. For example, a gene conversion method known as “SOEN”may be used to produce a chimeric accessory molecule gene which containsnucleotide segments derived from different chimeric accessory molecules.The methods for using this gene conversion method are well known in theart and have been described for example in Horton, R. M., Mol.Biotechnol., 3:93 (1995); Ali, S. A. and A. Steinkasserer,Biotechniques, 18:746 (1995); Vilardaga, J. P., E. Di Paolo, and A.Bollen, Biotechniques, 18:604 (1995); Majumder, K., F. A. Fattah, A.Selvapandiyan, and R. K. Bhatnagar, PCR. Methods Appl., 4:212 (1995);Boles, E. and T. Miosga, Curr. Genet. 28:197 (1995); Vallejo, A. N., R.J. Pogulis, and L. R. Pease, PCR. Methods Appl., 4:S123 (1994); Henkel,T. and P. A. Baeuerle, Anal. Biochem., 214:351 (1993); Tessier, D. C.and D. Y. Thomas, Biotechniques, 15:498 (1993); Morrison, H. G. and R.C. Desrosiers, Biotechniques, 14:454 (1993); Cadwell, R. C. and G. F.Joyce, PCR. Methods Appl., 2:28 (1992); and, Stappert, J., J. Wirsching,and R. Kemler, Nucleic Acids Res., 20:624 (1992). Alternatively, one ofordinary skill in the art will understand that site-directed mutagenesismay be used to introduce changes into a particular nucleotide sequenceto directly produce or indirectly be used to produce a chimericaccessory molecule gene of the present invention. For example, themutagen kit provided by BioRad Laboratories may be used together withthe methods and protocols described within that kit to produce thedesired changes in the nucleotide sequence. These methods wereoriginally described by Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492(1985) and Kunkel et al., Meth. Enzol. Mol., 154:367-382 (1987). Byusing the site directed mutagenesis protocols described herein and knownwithin the art, a skilled investigator may induce individual nucleotidechanges which result in an altered amino acid sequence or which preservean amino acid sequence but introduce a desired restriction enzymerecognition sequence into the gene. This new restriction endonucleaserecognition site may then be used to cut the gene at that particularpoint and use it to a gene or segment of another accessory moleculeligand gene. In addition to these methods, one of ordinary skill in theart will understand that an entire chimeric accessory molecule ligandgene may be synthesized using synthetic methods known in the art. Thismethodology only requires that the skilled artesian generatingnucleotide sequence of a chimeric accessory molecule ligand gene andprovide that sequence to a company which is capable of synthesizing sucha gene.

B. Genetic Constructs

The present invention contemplates the use of accessory molecule ligandgenes or chimeric accessory molecule ligand genes which are present invarious types of genetic vectors. A genetic vector refers to a DNAmolecule capable of autonomous replication in a cell into which anotherDNA segment can be inserted to cause the additional DNA segments toreplicate. Vectors capable of expressing genes contained in that vectorare referred to as “expression vectors.” Thus, the genetic vectors andexpression vectors of the present invention are recombinant DNAmolecules which comprise at least two nucleotide sequences not normallyfound together in nature.

The genetic vectors useful in the present invention contain an accessorymolecule ligand gene which encodes an accessory molecule ligand which isoptionally operatively linked to a suitable transcriptional ortranslational regulatory nucleotide sequence, such as one derived from amammalian, microbial, viral, or insect gene. Such regulatory sequencesinclude sequences having a regulatory role in gene expression, such as atranscriptional promoter or enhancer, an operator sequence to controltranscription, a sequence encoding a ribosomal binding site within themessenger RNA and appropriate sequences which control transcription,translation initiation or transcription termination.

Particularly useful regulatory sequences include the promoter regionsfrom various mammalian, viral, microbial, and insect genes. The promoterregion directs an initiation of transcription of the gene and causestranscription of DNA through and including the accessory molecule ligandgene. Useful promoter regions include the promoter found in the RousSarcoma Virus (RSV)—long terminal repeat (LTR), human cytomegalovirus(HCMV) enhancer/promoter region lac promoters, and promoters isolatedfrom adenovirus, and any other promoter known by one of ordinary skillin the art would understand to be useful for gene expression ineukaryotes, prokaryotes, viruses, or microbial cells. Other promotersthat are particularly useful for expressing genes and proteins withineukaryotic cells include mammalian cell promoter sequences and enhancersequences such as those derived from polyoma virus, adenovirus, simianvirus 40 (SV40), and the human cytomegalovirus. Particularly useful arethe viral early and late promoters which are typically found adjacent tothe viral origin of replication in viruses such as the SV40. Examples ofvarious promoters which have been used in expression vectors have beendescribed by Okiama and Berg (Mol. Cell. Biol. 3:280, 1983), the pMLSVNSV40 described by Kossman et al., Nature 312:768 (1984). One of ordinaryskill in the art will understand that the selection of a particularuseful promoter depends on the exact cell lines and the other variousparameters of the genetic construct to be used to express the accessorymolecule ligand gene or the chimeric accessory molecule ligand genewithin a particular cell line. In addition, one of ordinary skill in theart will select a promoter which is known to express genes in the targetcell at a sufficiently high level to be useful in the present invention.

The genetic vectors and expression vectors of the present inventionoptionally contain various additional regulatory sequences includingribosome binding sites which allow the efficient translation of themessenger RNA produced from an expression vector into proteins, the DNAsequence encoding various signals peptides which may be operativelylinked to the accessory molecule ligand gene or the chimeric accessorymolecule ligand gene. The signal peptide, if present, is expressed as aprecursor amino acid which enables improved extracellular secretion oftranslation fusion polypeptide.

The genetic constructs contemplated by the present invention thereforeinclude various forms of accessory molecule ligand genes described abovewhich are operatively linked to either a promoter sequence or a promoterand enhancer sequence and also operatively linked to a polyadenylationsequence which directs the termination and polyadenylation of messengerRNA. It is also contemplated that the genetic constructs of the presentinvention will contain other genetic sequences which allow for theefficient replication and expression of that construct within thedesired cells. Such sequence may include introns which are derived fromnative accessory molecule ligand genes or, for example, from a virusgene.

The present invention also contemplates gene therapy vectors which areable to directly infect mammalian cells so as to introduce the desiredaccessory molecule ligand gene or chimeric accessory molecule ligandgene into that cell. These gene therapy vectors are useful for directlyinfecting cells which have been isolated from an animal or patient, orcan be directly introduced into an animal or patient and therebydirectly infect the desired cell within that animal or patient.

Many types of gene therapy vectors which are able to successfullytransfer genes and cause the expression of desired foreign DNA sequenceshave been developed and described in the literature. For example, thearticle entitled “Gene Transfer Vectors for Mammalian Cells” in CurrentComm. Mol. Biol., Cold Springs Harbor Laboratory, New York (1987).Further, naked DNA can be physically introduced into eukaryotic cellsincluding human cells by transvection using any number of techniquesincluding calcium phosphase transfection (Berman et al., Proc. Natl.Acad. Sci. USA, 81:7176 (1984)), DEAE-Dextran Transfection, protoplastfusion (Deans et al., Proc. Natl. Acad. Sci. USA, 81:1292 (1984)),electroporation, liposome fusion, polybrene transfection and direct genetransfer by laser micropuncture of the cell membrane. In addition, oneof ordinary skill in the art will understand that any technique which isable to successfully introduce the DNA into a cell in such a manner asto allow it to integrate into the genome of a cell and allow theexpression of the desired gene would be useful in the present invention.

Specifically, gene therapy vectors which utilize recombinant infectiousvirus particles for gene delivery have been widely described. See, forexample, Brody, S. L. and R. G. Crystal, Ann. N. Y. Acad. Sci., 716:90(1994); Srivastava, A., Blood. Cells, 20:531 (1994); Jolly, D., CancerGene Ther., 1:51 (1994); Russell, S. J., Eur. J. Cancer, 30A:1165(1994); Yee, J. K., T. Friedmann, and J. C. Burns, Methods Cell Biol.,43 Pt A:99 (1994); Boris-Lawrie, K. A. and H. M. Temin, Curr. Opin.Genet. Dev., 3:102 (1993); Tolstoshev, P., Annu. Rev. Pharmacol.Toxicol., 33:573 (1993); and, Carter, B. J., Curr. Opin. Biotechnol.,3:533 (1992). The present invention contemplates the use of gene therapyvectors to carry out the desired methodology of the present invention byintroducing a gene encoding an accessory molecule ligand gene or achimeric accessory molecule ligand gene into the cell. Many viralvectors have been defined and used as gene therapy vectors and includevirus vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated viruses, and retroviruses. One of ordinary skill in theart will understand that useful gene therapy vectors are vectors whichare able to directly introduce into the target cells the DNA whichencodes the accessory molecule ligand and allow that DNA to persist inthe cell so as to express the accessory molecule ligand in the desiredmanner within the cell.

The gene therapy vectors of the present invention are useful forintroducing accessory molecule ligand genes into a variety of mammaliancells including human cells. The particular cells infected by the genetherapy vector will depend on the various specifics of the vector andsuch vectors can be used to introduce the accessory molecule ligandgenes of the present invention into hematopoietic or lymphoid stemcells, antigen presenting cells, embryonic stem cells, and other cellswhich are capable of presenting antigen within the immune systemincluding cells which have CD40 on their surface. Further, such genetherapy vectors are able to introduce a gene encoding an accessorymolecule ligand gene into a human neoplastic cell such as a lymphoma,leukemia, AML, CLL, CML, AMML, CMML, breast cancer, lung cancer, ovariancancer or any tumor capable of acting as antigen presenting cells orcells which can stimulate bystander antigen presenting cells. Further,the contemplated gene therapy vectors may be used to introduce theaccessory molecule ligand genes of the present invention into cellswhich have been engineered to make those cells capable of presentingantigen to the immune system.

III. Cells Containing Genetic Constructs Encoding an Accessory MoleculeLigand or Chimeric Accessory Molecule Ligand

The present invention also contemplates various cells which contain thegenetic constructs of the present invention. These cells contain theconstructs which encode the accessory molecule ligand gene and thuscontain the various genetic elements described in Section II.B. above.These cells may be microbial cells, eukaryotic cells, insect cells, andvarious mammalian cells including human cells. In preferred embodimentsof the present invention, these cells include various neoplastic cellsincluding human neoplastic cells. These neoplastic cells may be of anycell type and include cells of the immune system, and other blood cells.Particularly preferred are any neoplastic cells which may function as anantigen presenting cells within the immune system or which may stimulatebystander antigen presenting cells by expression of a transgenicaccessory cell molecule of the present invention. Typically theseneoplastic which are able to function to present antigen to the immunesystem have or have had an accessory molecule, such as the CD40molecule, on the cell surface. Generally, these cells are naturallycapable of presenting antigen to the immune system, but the presentinvention also contemplates the introduction of accessory moleculeligand genes into a cell which is not naturally able to present antigento the immune system but which has been genetically engineered to makethat cell capable of presenting antigen to the immune system. Typically,these cells include various known cell types such as monocytes,macrophages, B cells, Langerhans cells, interdigitating cells,follicular dendritic cells or Kupffer cells and the like which havebecome neoplastic. In addition, the present invention also contemplatescells from various carcinomas, breast, ovarian and lung cancers whichcontain the genetic constructs described herein. In other preferredembodiments, an accessory molecule ligand gene of the present inventionis placed into cells which may be injected into a treatment site such asa tumor bed or joint. For example, the accessory molecule ligand gene ofthe present invention may be inserted into a fibroblast cell and theaccessory molecule ligand expressed on the surface of that cell. Thefibroblasts are then injected into the treatment site and cause thedesired immuno effect due to the presence of the accessory moleculeligand on the surface of those cells. These cells stimulate other immunecells present in that treatment site (bystander cells). This processthen results in the desired effect on the immune system.

IV. Methods Utilizing Genetic Vectors and Constructs Containing anAccessory Molecule Ligand Gene

The present invention contemplates methods of altering theimmunoreactivity of human cells using a method which includesintroducing a gene encoding an accessory molecule ligand gene into thehuman cells so that the accessory molecule ligand encoded by that geneis expressed on the surface of those cells. The present invention isuseful for any human cells which participate in an immune reactioneither as a target for the immune system or as part of the immune systemwhich responds to the foreign target. A large variety of methods arecontemplated in which the final result is that the accessory moleculeligand gene is introduced into the desired cells. These methods includeex vivo methods, in vivo methods and various other methods which involveinjection of DNA, genetic vectors or gene therapy vectors into theanimal or human, including injection directly into the tumor bed presentin any animal or human.

Ex vivo methods are contemplated wherein the cells into which theaccessory molecule ligand gene is to be introduced are isolated from theanimal or patient and then the gene is introduced into those isolatedcells using suitable methods. Examples of useful ex vivo methods havebeen described for example by Raper, S. E., M. Grossman, D. J. Rader, J.G. Thoene, B. J. Clark, D. M. Kolansky, D. W. Muller, and J. M. Wilson,Ann. Surg., 223:116 (1996); Lu, L., R. N. Shen, and H. E. Broxmeyer,Crit. Rev. Oncol. Hematol., 22:61 (1996); Koc, O. N., J. A. Allay, K.Lee, B. M. Davis, J. S. Reese, and S. L. Gerson, Semin. Oncol., 23:46(1996); Fisher, L. J. and J. Ray, Curr. Opin. Neurobiol., 4:735 (1994);and, Goldspiel, B. R., L. Green, and K. A. Calis, Clin. Pharm., 12:488(1993). D. Dilloo et al., in Blood 90:1927-1933 (1997), describe amethod, using CD40L-activated cells, for treating B-acute lymphoblasticleukemia (ALL). They cocultured leukemia cells with fibroblasts infectedwith a retroviral vector encoding CD40L, then injected the cell mix intomice. Such an approach, if taken in humans, would differ from thatcontemplated here in that the therapeutic cells are stimulated in vitro,by another cell line expressing the accessory molecule ligand. Schultze,J. L. et al., in Blood 89: 3806-3816 (1997), describe a method forstimulating T-TILs (tumor-infiltrating T cells) cytotoxic for follicularlymphoma (FL) cells by exposing them, in vitro, to FL B cells which werepreviously cultured with CD40L-expressing fibroblasts. They propose anadoptive immunotherapy in which T-TILS stimulated in this manner aretransfused into patients. This method also requires in vitrostimulation, of the cells to be transfused, with another cell lineexpressing an accessory molecule.

Following the introduction of the gene, including any optional steps toassure that the accessory molecule ligand gene has been successfullyintroduced into those isolated cells, the isolated cells are introducedinto the patient either at a specific site or directly into thecirculation of the patient. In preferred embodiments of the presentinvention, cell surface markers, including molecules such tumor markersor antigens identify the cells are used to specifically isolate thesemolecules from the patient. One of ordinary skill in the art willunderstand that such isolation methods are well known and include suchmethodologies as fluorescence activated cell sorting (FACS),immunoselection involving a variety of formats including panning,columns and other similar methods.

The present invention also contemplates introducing the accessorymolecule ligand gene into the desired cells within the body of an animalor human patient without first removing those cells from the patient.Methods for introducing genes into specific cells in vivo, or within thepatient's body are well known and include use of gene therapy vectorsand direct injection of various genetic constructs into the animal orpatient. Examples of useful methods have been described by Danko, I. andJ. A. Wolff, Vaccine, 12:1499 (1994); Raz, E., A. Watanabe, S. M. Baird,R. A. Eisenberg, T. B. Parr, M. Lotz, T. J. Kipps, and D. A. Carson,Proc. Natl. Acad. Sci. U. S. A., 90:4523 (1993); Davis, H. L., R. G.Whalen, and B. A. Demeneix, Hum. Gene Ther., 4:151 (1993); Sugaya, S.,K. Fujita, A. Kikuchi, H. Ueda, K. Takakuwa, S. Kodama, and K. Tanaka,Hum. Gene Ther., 7:223 (1996); Prentice, H., R. A. Kloner, Y. Li, L.Newman, and L. Kedes, J. Mol. Cell Cardiol., 28:133 (1996); Soubrane,C., R. Mouawad, 0. Rixe, V. Calvez, A. Ghoumari, O. Verola, M. Weil, andD. Khayat, Eur. J. Cancer, 32A:691 (1996); Kass-Eisler, A., K. Li, andL. A. Leinwand, Ann. N. Y. Acad. Sci., 772:232 (1995); DeMatteo, R. P.,S. E. Raper, M. Ahn, K. J. Fisher, C. Burke, A. Radu, G. Widera, B. R.Claytor, C. F. Barker, and J. F. Markmann, Ann. Surg., 222:229 (1995);Addison, C. L., T. Braciak, R. Ralston, W. J. Muller, J. Gauldie, and F.L. Graham, Proc. Natl. Acad. Sci. U. S. A., 92:8522 (1995); Hengge, U.R., P. S. Walker, and J. C. Vogel, J. Clin. Invest., 97:2911 (1996);Felgner, P. L., Y. J. Tsai, L. Sukhu, C. J. Wheeler, M. Manthorpe, J.Marshall, and S. H. Cheng, Ann. N. Y. Acad. Sci., 772:126 (1995); and,Furth, P. A., A. Shamay, and L. Hennighausen, Hybridoma, 14:149 (1995).In a typical application, a gene therapy vector containing an accessorymolecule ligand gene is introduced into the circulation or at alocalized site of the patient to allow the gene therapy vector tospecifically infect the desired cells. In other preferred embodimentsthe gene therapy vector is injected directly into the tumor bed presentin an animal which contains at least some of the cells into which theaccessory molecule ligand gene is to be introduced.

The present invention also contemplates the direct injection of DNA froma genetic construct which has a promoter and accessory molecule ligandgene followed by a polyadenylation sequence into a patient or animal.Examples of such useful methods have been described by Vile, R. G. andI. R. Hart, Ann. Oncol., 5 Suppl 4:59 (1994). The genetic construct DNAis directly injected into the muscle or other sites of the animal orpatient or directly into the tumor bed of the animal or patient.Alternatively, DNA from a genetic construct containing at least anaccessory molecule ligand gene is used and directly injected into theanimal.

In preferred embodiments of the present invention, the immune reactionor response of a human patient or animal is altered by introducing theaccessory molecule ligand gene into cells, including human cells whichhave an accessory molecule present on the cell surface. Such cellsinclude human cells, human antigen presenting cells and optionally thesecells may be neoplastic antigen presenting cells which have the capacityto express the accessory molecule on the surface of the cell or cellswhich are capable of stimulating. In some embodiments, the amount ofaccessory molecule present on the surface of the cells into which theaccessory molecule ligand gene is to be introduced is very small andsuch small amounts of the accessory molecule may result fromdown-regulation of that accessory molecule on the surface of such cells.In some embodiments, the cells into which the accessory molecule ligandgene is introduced have at least low levels of the CD40 molecule presenton the cell surface or are derived from cells which did express the CD40ligand molecule on the cell surface but have reduced or eliminated thatexpression.

The preferred methods of altering the immunoreactivity of a particularcell are applicable to mammalian cells including human cells. Thesehuman cells may include neoplastic human cells such as human lymphomas,leukemias, and other malignancies including breast, lung and ovariancancers. In some preferred embodiments the cells are normal antigenpresenting cells of a human patient such as monocytes, macrophages, Bcells, Langerhans cells, interdigitating cells, follicular dendriticcells, Kupffer cells, and other similar cells. In preferred embodiments,the cells are lymphocytes which acquire altered immunoreactivity whenthe accessory molecules of the present invention are introduced intothose cells. In other preferred embodiments, the cells may be neoplasticor normal cells which are capable of stimulating bystander antigenpresenting cells when the accessory molecule ligand genes of the presentinvention are introduced into these cells. The present invention alsocontemplates that cells which are not naturally capable of presentingantigen to the immune system may be genetically engineered to introducethe genes encoding the molecules required for antigen presentation,including genes encoding an accessory molecule, and thus allow thesecells to act as artificial antigen presenting cells. The accessorymolecule ligand gene may then be introduced into these artificialantigen presenting cells. Various tests are well known in the literatureto determine whether a particular cell is able to function as an antigenpresenting cell, such as cell proliferation or the production oflymphokines and therefore this aspect of the present invention may beeasily determined.

In addition to the above normal human cells, the present invention alsocontemplates introducing the accessory molecule ligand gene into variousneoplastic or malignant cells which optionally are antigen presentingcells. Such human neoplastic cells which are contemplated includeleukemias, lymphomas, AML, AMML, or CMML, CML, CLL and any neoplasticcell which is capable of stimulating bystander antigen presenting cellswhen an accessory molecule ligand is introduced into that cell. Alsocontemplated are neoplastic cells such as a breast, ovarian or lungcancer cell which is capable of or is engineered to act as an antigenpresenting cell. However, the present immunomodulation also applicableto other malignancies not specifically identified and thus would includeany tumor of any cell capable of presenting antigen within the animal orhuman immune system or any cell which is capable of acting as an antigenpresenting cell or capable of stimulating bystanding antigen presentingcells after an accessory molecule ligand gene has been introduced intothose cells. Generally these antigen presenting cells have accessorymolecules on the surface of the cells.

The present methods of altering the immunoreactivity of a human oranimal cell contemplate the introduction of an accessory molecule ligandgene into the cells for which altered immunoreactivity is desired. Thegenes useful in the present invention include the wide range ofaccessory molecule ligand genes and chimeric accessory molecule ligandgenes identified above and in preferred embodiments include at least aportion of the murine CD40 ligand gene. In particularly preferredembodiments, the accessory molecule ligand gene introduced into thecells using the methods of the present invention is selected tocorrespond to the accessory molecule present on the surface of the cellsfor which altered immunoreactivity is desired. In one particularapplication of the present invention, the immunoreactivity of a cellwhich expresses the CD40 molecule on the cell surface would beaccomplished by introducing the gene which encodes the CD40 ligandmolecule and more preferably the murine CD40 ligand molecule.

The present invention also contemplates altering the immunoreactivity ofhuman or animal cells by introducing an accessory molecule ligand genewhich is a chimeric accessory molecule ligand gene into the cell. Thevarious useful chimeric accessory molecule ligand genes were identifiedabove and could include a wide variety of molecules and allow the uniqueproperties of those chimeric accessory molecule ligand genes to beutilized to alter the immunoreactivity of the target cells. In preferredembodiments, useful chimeric accessory molecule ligand genes are geneswhich encode at least a portion of the accessory molecule ligand whichis capable of binding the accessory molecule present on the surface ofthe cells for which altered immunoreactivity is desired.

The methods of the present invention for altering the immunoreactivitycontemplate the use of genetic vectors and genetic constructs includinggene therapy vectors which encode an accessory molecule ligand andtherefore contain an accessory molecule ligand gene. Typically, thegenetic vectors and genetic constructs including the gene therapyvectors of the present invention have a promoter which is operativelylinked to the accessory molecule ligand gene followed by apolyadenylation sequence. In other embodiments, the only requirement isthat the genetic vectors, genetic constructs, and gene therapy vectorsof the present invention contain the accessory molecule ligand gene orthe chimeric accessory molecule ligand gene.

V. Methods of Treating Neoplasia

The present invention also contemplates methods of treating humanneoplasia comprising inserting into a human neoplastic cell a gene whichencodes an accessory molecule ligand so that the accessory moleculeligand is expressed on the surface of the neoplastic cells. The presentinvention contemplates treating human neoplasia both in vivo, ex vivoand by directly injecting various DNA molecules containing a gene whichencodes an accessory molecule ligand into the patient. However, at aminimum, the present methods for treating human neoplasia involveinserting the gene encoding the accessory molecule ligand into theneoplastic cells in such a way as to allow those neoplastic cells toexpress the accessory molecule ligand on the cell surface. Theexpression of the accessory molecule ligand gene in these neoplasticcells modulates the immune system to cause the neoplasia to be reducedor eliminated.

In a preferred method of treating human neoplasia, the method furthercomprises the steps of first obtaining the human neoplastic cells from ahuman patient and then inserting into the isolated human neoplasticcells a gene which encodes an accessory molecule ligand so that theaccessory molecule ligand is expressed on the surface of the neoplasticcells. The human neoplastic cells having the accessory molecule ligandon the surface of that cell are then infused back into the humanpatient. One of ordinary skill in the art will understand that numerousmethods are applicable for infusing the altered human neoplastic cellscontaining the gene encoding the accessory molecule ligand back into thepatient and that these methods are well known in the art.

The contemplated methods of treating human neoplasia are applicable to awide variety of human neoplasias including lymphomas, leukemias, andother malignancies. In preferred embodiments the human neoplasia is aneoplasia: which involves the antigen presenting cells of the humanimmune system and includes monocytes, macrophages, B cells, Langerhanscells, interdigitating cells, follicular dendritic cells, Kupffer cells,and the like. In other preferred embodiments, the human neoplasia is aleukemia, a lymphoma, AML, AMML, CMML, CML or CLL, lung cancer, breastcancer, ovarian cancer and other similar neoplasias.

The genetic vectors, genetic constructs and gene therapy vectors usefulin the methods of treating human neoplasia of the present invention havebeen disclosed above and include constructs in which a promoter isoperatively linked to the accessory molecule ligand gene or the chimericaccessory molecule ligand gene which is in turn operatively linked to apolyadenylation sequence. The methods of treating human neoplasiacontemplate the use of genetic constructs, genetic vectors and genetherapy vectors as described in this specification. In addition, thepresent invention contemplates the use of DNA which contains at least agene encoding an accessory molecule ligand gene. This gene may or maynot contain a promoter and other regulatory sequences.

In preferred embodiments of the present invention, the cells comprisingthe human neoplasia are located in at least one defined site termed atumor bed within the human patient. This tumor bed typically containsthe tumor or neoplastic cell together with a number of other cells whichare associated with the tumor or neoplastic cells. The present inventioncontemplates methods of treating such human neoplasia present in a tumorbed by injecting into the tumor bed of the patient, a gene which encodesan accessory molecule ligand so that the accessory molecule ligand isexpressed on the surface of the tumor cells thereby causing the cells toparticipate in an immune reaction. The gene which encodes the accessorymolecule ligand may be present as part of a gene therapy vector, geneticconstruct or genetic vector.

In preferred embodiments, the accessory molecule ligand gene is achimeric accessory molecule ligand gene which has at least a portion ofthe murine CD40 ligand gene is used. In other preferred embodiments, theaccessory molecule ligand encoded is capable of binding an accessorymolecule present on the human neoplasia to be treated.

The various gene therapy vectors used in the treatment methods of thepresent invention include vectors which are capable of directlyinfecting human cells. Such vectors have been described in theliterature and are readily adaptable to the methods described in thepresent invention.

The present invention contemplates the use of any type of gene therapyincluding the methods of Raper, S. E. et al., Ann. Surg., 223:116(1996); Lu, L. et al., Crit. Rev. Oncol. Hematol., 22:61 (1996); Koc, O.N. et al., Semin. Oncol., 23:46 (1996); Fisher, L. J. et al., Curr.Opin. Neurobiol., 4:735 (1994); Goldspiel, B. R. et al., Clin. Pharm.,12:488 (1993); Danko, I. et al., Vaccine, 12:1499 (1994); Raz, E. etal., Proc. Natl. Acad. Sci. U.S.A., 90:4523 (1993); Davis, H. L. et al.,Hum. Gene Ther., 4:151 (1993); Sugaya, S. et al., Hum. Gene Ther., 7:223(1996); Prentice, H. et al., J. Mol. Cell Cardiol., 28:133 (1996);Soubrane, C. et al., Eur. J. Cancer, 32A:691 (1996); Kass-Eisler, A. etal., ann. N. Y. Acad. Sci., 772:232 (1995); DeMatteo, R. P. et al., Ann.Surg., 222:229 (1995); Addison, C. L. et al., Proc. Natl. Acad. Sci.U.S.A., 92:8522 (1995); Hengge, U. R. et al., J. Clin. Invest., 97:2911(1996); Felgner, P. L. et al., Ann. N. Y. Acad. Sci., 772:126 (1995);Furth, P. A., Hybridoma, 14:149 (1995); Yovandich, J. et al., Hum. GeneTher., 6:603 (1995); Evans, C. H. et al., Hum. Gene Ther., 7:1261.

VI. Methods of Vaccination

The present invention contemplates methods of vaccinating an animalagainst a predetermined organism comprising administering to that animala vaccine containing immunogenic animal antigens capable of causing animmune response in that animal against the desired organism togetherwith a vector containing a gene encoding an accessory molecule ligand.The present invention also contemplates methods of vaccinating an animalwhich include administering the genes which encode the immunogenicantigen capable of causing a desired immune response or altering theimmune response to a particular antigen together with a vectorcontaining a gene including the accessory molecule ligand gene. In thisparticular embodiment, the vector or vectors introduced encode theimmunogenic antigens desired and the desired accessory molecule ligand.The present invention also contemplates that the gene or genes encodingthe immunogenic peptide or peptides may be present on the same vector asis the gene or genes encoding the accessory molecule ligand.

The vaccination methods of the present invention are general in thatthey may be used to produce a vaccination against any predeterminedorganism, such as a virus, a bacteria, a fungus or other organism. Inaddition, the present vaccination methods may be used to produce animmune response against a neoplastic cell.

In other preferred embodiments, the vaccination methods of the presentinvention utilize a genetic vector, a genetic construct or a genetherapy vector which contains an accessory molecule ligand gene which isa chimeric accessory molecule ligand gene. That chimeric accessorymolecule ligand gene preferably contains at least a portion of themurine CD40 ligand gene. In other preferred embodiments, the vaccinationmethod utilizes a DNA molecule which encodes at the minimum theaccessory molecule ligand gene or a chimeric accessory molecule ligandgene. This particular DNA may or may not include a promoter sequencewhich directs the expression of the accessory molecule ligand gene.

The present invention also contemplates that the vaccination method mayutilize a genetic vector which is capable of expressing an accessorymolecule ligand within a particular cell or organism together with avector which is capable of expressing at least a single polypeptide froman andovirus. This andovirus polypeptide may be expressed from the sameor different vector which expresses the accessory molecule ligand inthat cell. In this particular embodiment, the andovirus polypeptide isalso expressed in at least one cell type within the organism and servesto modulate the immune response found in response to this vaccinationprotocol.

The present invention also contemplates the introduction of an accessorymolecule ligand gene into cells which are present in the joints ofpatients with rheumatoid arthritis. In preferred embodiments, theaccessory molecule ligand gene introduced comprises at least a portionof the Fas ligand gene and upon expression the accessory ligand inducesthe cell death of cells expressing Fas on the cell surface. This processleads to the reduction of the destructive inflammatory process.

The following examples are provided to illustrate various aspects of thepresent invention and do not limit the scope of that invention.

VII. Methods of Treating Arthritis

The present invention also contemplates methods of treating arthritiscomprising inserting into a joint, cells which have been transformedwith an accessory molecule, such as the Fas ligand. In preferredembodiments, the expression of that accessory molecule ligand or thestability of that molecule on the surface of the cells has been altered.In these preferred embodiments, the accessory molecule ligand functionsin an enhanced manner to aid in the treatment of arthritis within thejoint. The present invention contemplates treating human arthritis bothin vivo, ex vivo, and by directly injecting various DNA moleculescontaining genes which encode the useful accessory molecule ligand intothe patients. Various useful protocols may be designed to rheumatoidarthritis including those described in the example section below.

The present invention contemplates the treatment of arthritis utilizingaccessory molecule ligand genes which may be chimeric accessory moleculeligand genes comprised of portions of that gene being derived from twodifferent accessory molecule ligand genes. In other embodiments, thechimeric accessory molecule ligands may be produced by utilizing domainsfrom the same accessory molecule ligand gene. The resulting chimericaccessory molecule ligands have an altered stability on the surface ofcells upon which they are expressed. This altered stability modulatesthe function of the immune system in the local environment around thecells in which these chimeric accessory molecule ligands are expressed.For example, in certain preferred embodiments, Fas ligand stability isaltered on the surface of cells within a joint of a patient sufferingfrom arthritis. This altered stability modulates the immune system andcauses the cells to be targeted for apoptosis and thus reducing theimmune response within the inflamed joint. In other embodiments, theaccessory molecule ligand genes described within are altered such thatthe resulting accessory molecule ligand has an altered stability andcauses an immunomodulatory effect which can be useful in the treatmentof arthritis.

The present invention contemplates in preferred embodiments thatchimeric accessory molecule ligands genes be utilized in the treatmentof arthritis. These chimeric accessory molecule ligand genes preferablycontain at least a portion of the Fas ligand gene Domain IV, whichcarries the effect or function for Fas ligand. In preferred embodiments,at least in the portion of that domain, is present which allows Fasligand to have its biologic effects. In other preferred chimericaccessory molecule ligands, those ligands contain domains from otheraccessory molecule ligand genes of the present invention or from adifferent domain of the same accessory molecule ligand. Particularlypreferred are Fas chimeric accessory molecule ligand genes made up onDomain IV of the human Fas ligand operatively linked with Domain III ofthe mouse Fas ligand. This particular combination results in more stableFas ligand and thus, by replacing Domain III of human Fas ligand withDomain III of the mouse ligand, the activity of the human Fas ligandgene is altered.

Alternatively, in other preferred embodiments, the murine Fas ligandgene is used to encode the murine Fas ligand on the surface of cells inplace of the human Fas ligand. The murine Fas ligand is more stable thanthe human Fas ligand and thus, alters the Fas ligand activity in thejoint. The resulting alter Fas ligand activity is useful in thetreatment of rheumatoid arthritis.

Further preferred embodiments include embodiments in which the effect orfunction present on Domain IV of the human Fas ligand is combined withother domains from other accessory molecule ligands. For example, CD70Domain III is more stable than Domain III of the human Fas ligand andthus the chimeric accessory molecule ligand made up of Domain III fromthe human CD70 and Domain IV of the Fas ligand together with othersupporting domains would be more stable. The increased stability leadsto increase Fas ligand activity. In other preferred embodiments, DomainIII of the Fas ligand is replaced with multiple copies of a domain ordomains. Such multiple copies of domains include domains made up of twoor more copies of other domains such as Domains III or I of the CD70molecule.

In other preferred embodiments, the present invention contemplatesaccessory molecule ligand genes, such as Fas ligand genes, in which acleavage site for matrix-metalloproteinase (MMP), have been removed fromthe accessory molecule ligand. MMP cleavage and recognition sites,charted in FIG. 28, are discussed in Smith, M. M. et al., Journal ofBiol. Chem. 270:6440-6449 (95) and Nagase, H., and G. B. Fields,Biopolymers (Peptide Science) 40:399-416 (96). In preferred embodiments,at least one MMP site has been removed from at least Domain III of theFas ligand gene. The removal of the MMP site from the Fas ligand genemakes the Fas ligand more stable and thus, more effective in thetreatment of arthritis.

In other preferred embodiments, chimeric accessory molecule ligand genesare comprised of portions of the human Fas ligand gene with otherdomains from other human accessory molecule ligands or domains fromaccessory molecules derived from other species. For example, the presentinvention contemplates the use of domains from CD40 ligand, CD70 ligand,CD30 ligand, TNF-related apoptosis inducing ligand (TRAIL), TNF-α aswell as mutants of human Fas ligand and murine Fas ligand. Production ofsuch chimeric accessory molecule ligands is easily accomplished bymanipulating and producing accessory molecular ligand genes which arechimeric and thus has portions derived from at least two differentaccessory module ligand genes.

EXAMPLES 1. Expression of Human and Mouse Accessory Molecule Ligand inHuman CLL Cells a. Construction of a Genetic Construct and Gene TherapyVector Containing a Human and Mouse Accessory Molecule Ligand Gene

Either the human accessory molecule ligand gene (human CD40 ligand) orthe murine accessory molecule ligand gene (murine CD40 ligand) wasconstructed utilizing the respective human and murine genes. Each ofthese genes was cloned in the following manner.

i. Murine CD40-L Cloning

Total RNA was isolated using the RNA STAT-60 kit (Tel-Test “B” Inc.,Friendswood, Tex.) from 1×10⁷ B6 mouse splenocytes that were previouslyactivated for 8 hours with immobilized CD3-specific mAb. cDNA was thensynthesized with the Superscript cDNA synthesis kit (Gibco BRL, GrandIsland, N.Y.) using oligo-dT primers. The murine CD40 ligand (mCD40-L)gene was then amplified from the cDNA by PCR using the following mCD40-Lspecific primers. 5′-GTTAAGCTTTTCAGTCAGCATGATAGAA (SEQ ID NO: 26),5′-GTTTCTAGATCAGAGTTTGAGTAAGCC (SEQ ID NO: 27). The amplified mCD40-LPCR product was subcloned into the HindIII and Xbal sites of theeukaryotic expression vector pcDNA3 (Invitrogen, San Diego, Calif.). ADNA fragment encompassing the CMV promoter, mCD40-L gene, andpolyadenylation signal was released from this plasmid construct afterrestriction digestion with Bg1II and XhoI enzymes. This DNA fragment wasthen subcloned into the shuttle plasmid MCS(SK)pXCX2 (Spessot R, 1989,Virology 168:378) that was designated mCD40-L pXCX2. This plasmid wasused for adenovirus production as described below.

ii. Human CD40-L Cloning

A plasmid containing the gene for human CD40-L was used to produce thehuman CD40-L gene used herein. The sequence of this gene is availableand thus this source of the gene was used merely for convenience. SeeGenBank accession no. X67878. This plasmid was used for PCRamplification of the human CD40-L gene using the specific primers, senseprimer 5′ CCAAGACTAGTTAACACAGCATGATCGAAA 3′ (SEQ ID NO: 28) andantisense primer 5′ CCAATGCGGCCGCACTCAGAATTCAACCTG 3′ (SEQ ID NO: 29).

These primers contain flanking restriction enzyme sites for subcloninginto the eukaryotic expression plasmid pRc/CMV (Invitrogen). The PCRamplified CD40-L fragment was subcloned into the SpeI and NotI sites ofpRc/CMV and designated hCD40-L pRc/CMV. A BglII and XhoI fragmentencompassing the CMV promoter, hCD40-L gene, and polyadenylation signalwas then released from this plasmid and subcloned into the shuttleplasmid MCS(SK)pXCX2 as described above. This plasmid was designatedhCD40-L pXCX2. This plasmid was used for adenovirus production asdescribed below.

iii. Adenovirus Synthesis

Either mCD40-L pXCX2 or hCD40-L pXCX2 plasmids were co-transfected withpJM17 (Graham and Prevec, 1991, Methods in Molecular Biology, Vol 7)into 293 cells (American Type Culture Collection, Rockville, Md.) usingthe calcium phosphate method (Sambrook, Fritsch, and Maniatis, 1989,Molecular Cloning, A Laboratory Manual, 2nd edition, chapter 16:33-34).Isolated adenovirus plaques were picked and expanded by again infecting293 cells. High titer adenovirus preparations were obtained as described(Graham and Prevec, 1991, Methods in Molecular Biology, Vol 7), exceptfor the following modifications. The cesium chloride gradient used forconcentrating viral particles was a step gradient, with densities of1.45 g/cm³ and 1.2 g/cm³. The samples were spun in a SW41 rotor(Beckman, Brea, Calif.) at 25,000 rpm at 4° C. The viral band wasdesalted using a Sephadex G25 DNA grade column (Pharmacia, Piscataway,N.J.). The isolated virus was stored at 70° C. in phosphate bufferedsaline with 10% glycerol. The virus titer was determined by infecting293 cells with serial dilutions of the purified adenovirus and countingthe number of plaques formed. Viral titers typically ranged from 10¹⁰ to10¹² plaque forming units/ml (PFU/ml).

b. Introduction of a Murine and Human Accessory Molecule Ligand Geneinto CLL Cells and HeLa Cells

For adenovirus infection, 10⁶ freshly thawed and washed CLL cells orHeLa cells were suspended in 0.5 to 1 mL of culture medium for cultureat 37° C. in a 5% CO₂-in-air incubator. Adenovirus was added to thecells at varying multiplicity of infection (MOI), and the infected cellswere cultured for 48 hours, unless otherwise stated, before beinganalyzed for transgene expression.

c. Expression of an Accessory Molecule Ligand Gene in CLL Cells and HeLaCells

The CLL and HeLa cells which were infected with the adenovirus vectorcontaining either mouse or human CD40 ligand genes prepared in Example1b. were then stained with commercially available monoclonal antibodiesimmunospecific for either human or mouse CD40 ligand (Pharmingen, SanDiego, Calif.) using the manufacturer's directions. The CLL and HeLacells were washed in staining media (SM) consisting of RPMI-1640, 3%fetal calf serum and 0.05% sodium azide and containing propidium iodideand then analyzed on a FACScan (Becton Dickinson, San Jose, Calif.).Dead cells and debris were excluded from analysis by characteristicforward and side light scatter profiles and propidium iodide staining.Surface antigen expression was measured as the mean fluorescenceintensity ratio (MFIR). MFIR equals the mean fluorescence intensity(MFI) of cells stained with a specific FITC-conjugated MoAb, divided bythe MFI of cells stained with a control IgG-FITC. This method controlsfor the nonspecific increases in auto-fluorescence seen in larger, moreactivated cells.

The histograms, generated for the CLL cells and HeLa cells containingeither a genetic vector containing the human CD40 ligand gene or themurine CD40 ligand gene and the appropriate controls, are shown in FIG.3A-3D. The expression of both the murine and human accessory moleculeligand gene (CD40 ligand) in HeLa cells is shown in FIGS. 3A and 3B,respectively. The expression of the murine and human accessory moleculeligand in CLL cells is shown in FIGS. 3C and 3D. The expression of anaccessory molecule ligand gene in CLL cells and the expression of murineCD40 ligand on the surface of the CLL cells is shown in FIG. 3C. Thefailure of the human accessory molecule ligand to be expressed on thesurface of the CLL cells is shown in FIG. 3D.

FIG. 8 shows data from an experiment done to examine whether the CD4⁺ Tcells of CLL patients could be induced to express the accessory moleculeligand mRNA after CD3 ligation. An ELISA-based quantitative competitiveRT-PCR was used to measure CD40 ligand transcript levels. In thisexperiment, CD40 ligand and RNA transcribed from the CD40 ligand gene inCLL cells are compared with levels of CD40 ligand and RNA made in normaldonor cells, after induction by CD3 ligation. For CD3 activation, platecoats of CD3 mAb were made and incubated with plated CLL or normal donormononuclear cells for the indicated amount of time, after which cellswere analyzed for expression of surface antigens or CD154 RNA messagelevels. CLL or normal donor serum was added to the cells at thebeginning of the activation assay for examination of modulation of CD40ligand surface expression.

For quantitative CD154 RT-PCR ELISA, total RNA was extracted andcompetitor RNA was generated from the insert containing CD40 ligand(CD154) cDNA. Varying amounts of competitor RNA were added to separatewells of isolated total RNA that subsequently were converted into cDNA.CD3 activation, ELISAs and PCR reactions were performed as described inCantwell, M. et al., Nature Medicine 3:984-989 (1997). Biotinylated PCRproducts were captured onto microtiter plates (Becton Dickinson, Oxnard,Calif.) coated with streptavidin (Sigma), and incubated. The plate wastreated with NaOH to remove the sense strands and subsequently washed.The DNA was then hybridized with either wild-type gene-specific orcompetitor-specific oligonucleotides. Using terminal transferase, eachprobe was labeled with a molecule of digoxigenin-11-dideoxyUTP(Boehringer Mannheim). The plate was incubated and washed with HYBEbuffer and blocking buffer, then peroxidase-conjugated anti-digoxigeninantibody (150 U/ml; Boehringer Mannheim) in blocking buffer was added.TMB (tetramethylbenzidine) and peroxidase (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) were added for color development, andoptical densities were measured at 450 nm and Deltasoft II(Biometallics, Princeton, N.J.) was used for data analysis.

Standard curves plotting the moles of RNA product versus the opticaldensity were made for the standard cDNA reactions. The equationsdescribing these standard curves were then used to calculate the molesof wild-type or competitor DNA present in the unknown PCR reactionsbased on the optical densities obtained in the ELISA readings. The ratioof the quantity of wild-type DNA to the amount of competitor DNA wasthen plotted against the known quantity of competitor RNA added in theinitial samples. The ratio of 1 was taken for the extrapolation of theamount of unknown moles of target RNA in the sample (a ratio of 1 meansthe amount of target RNA versus competitor RNA are equal). The moleculesof target RNA per CD4 cell was then calculated based on the followingformula: [(moles target CD154 RNA)×(6×10²³ molecules/mole)×(dilutionfactor of test RNA)]/(% of CD4 T cells in total cell population).

The upper graph in FIG. 8 shows that T cells of patients with CLL do notexpress detectable CD40 ligand after CD3 ligation. CD40 ligand RNA isproduced, but it is not stable. Although both CD40 ligand and CD40ligand RNA are expressed in normal donor T cells (lower graph), thelevels of neither the protein or RNA are stably maintained.

FIG. 9 shows a time course for surface expression of CD40 ligand.Expression reached a peak level at 48 hours after infection andpersisted at high levels for at least 6 days thereafter. In thisexperiment, CLL B cells were infected with a gene therapy vectorcontaining an accessory molecule ligand, at a MOI of 1000 at time zero,and then assessed by flow cytometry at various times thereafter. At eachtime point listed on the abscissa, the proportions of viable CLL B cellsthat expressed detectable CD154 are indicated by the vertical barscorresponding to the percentage scale depicted on the right-handordinate.

d. Function of the Human and Murine Accessory Molecule Ligands i.Induction of CD80 and CD54 on Cells Containing a Gene Therapy VectorEncoding an Accessory Molecule

The CLL cells infected with the murine accessory molecule ligand geneprepared in Example 1b. were then cultured in tissue culture plates. TheCLL cells were then analyzed using multiparameter FACS analysis todetect induction of CD80 and CD54 expression using fluroesceinisothiocyanate-conjugated monoclonal antibodies immunospecific for eachof these respective surface antigens. Non-infected CLL cells were usedas a control. The cells were subjected to the appropriate FACS analysisand histograms were generated. CD80 mAb was obtained from Dr. EdwardClark and CD54 mAb was purchased from CALTAG Inc. The CD80 wasconjugated using standard methods which have been described in Kipps etal., Laboratory Immunology II, 12:237-275 (1992).

The results of this analysis are shown in FIG. 4A-4D. FIGS. 4A-4Bcompare the amount of CD54 expression in CLL cells which have not beentransfected (FIG. 4A) or CLL cells into which a gene therapy vectorcontaining the murine CD40 ligand gene was introduced (FIG. 4B). Theshaded graph indicates the isotype control for FACS staining and theopen graph indicates the cells stained with the anti-CD54 antibody.These results show that the level of expression of CD54 is increased inCLL cells into which the gene therapy vector containing the murine CD40ligand was introduced.

FIGS. 4C and 4D compare the amount of CD80 expression in CLL cells whichhave not been transfected (FIG. 4C) or CLL cells into which a genetherapy vector containing the murine CD40 ligand gene was introduced(FIG. 4D). The shaded graph indicates the isotype control for FACSstaining and the open graph indicates the cells stained with theanti-CD80 antibody. These results show that the level of expression ofCD80 is increased in the CLL cells into which the gene therapy vectorcontaining the murine CD40 ligand was introduced.

In an additional experiment, CLL cells infected with a gene therapyvector containing the murine accessory molecule ligand gene wereevaluated by flow cytometry for induced expression of not only CD54 andCD80, but also CD86, CD58, CD70 and CD95. Fluorescein-conjugated mAbspecific for human CD54 and CD70 were purchased from CALTAG.Fluorescein-conjugated mAb specific for human CD27, CD58, CD80, CD86, orCD95, and phycoerythrin-conjugated mAb specific for human or mouse CD40ligand, were obtained from PharMingen. Shaded histograms representstaining of CLL B cells with FITC-conjugated isotype nonspecific mAb. Incontrast to uninfected CLL cells (FIG. 10, thin-lined histograms), orAd-lacZ-infected CLL cells (data similar to that obtained withuninfected cells, but not shown), CLL cells infected with the adenovirusvector encoding the CD40 ligand (CD154) expressed high levels of CD54(FIG. 10, top left), CD80 (FIG. 10, top middle), CD86 (FIG. 10, topright), CD58 (FIG. 10, bottom left), CD70 (FIG. 10, bottom middle), andCD95 (FIG. 10, bottom right). On the other hand, CD40 ligand-CLL (CD154CLL) expressed significantly lower levels of both surface membrane CD27(FIG. 11A, thick-lined histogram) and soluble CD27 (FIG. 11B) thanuninfected (FIG. 11A, thin-lined histogram) (P<0.01, Bonferroni t-test)or Ad-lacZ-infected CLL cells (data similar to that obtained withuninfected cells, but not shown). In the experiment shown in FIG. 11A,the CLL B cells were examined for expression of CD27 via flow cytometry,three days after infection. Shaded histograms represent staining of CLLB cells with FITC-conjugated isotype control mAb. In FIG. 11B, cell-freesupernatants were collected, after the infection or stimulation of CLL Bcells, for 72 hours and tested for the concentration of human CD27 byELISA. The reduced expression of CD27 (FIG. 11B) is similar to thatnoted for leukemia B cells stimulated via CD40 cross-linking with mAbG28-5 presented by CD32-expressing L cells, as described in Rassenti, L.Z. and T. J. Kipps, J. Exp. Med. 185:1435-1445.

ii. Allogeneic T Cell Responses to CLL Cells into which a GeneticTherapy Vector Containing a Murine CD40 Ligand Gene has been Introduced

The ability of CLL cells which have been infected with a gene therapyvector containing the murine CD40 ligand gene to stimulate allogeneic Tcells (i.e., from another individual) was analyzed using cellproliferation assays. Briefly, the test cells were co-cultured with thegenetic therapy vector containing the lac-Z gene or the murine CD40ligand gene at a multiplicity of infection of 1,000 in the presence ofIL-4 at a concentration of 10 ng/ml. In other samples, the CLL cellswere stimulated with MOPC21 (a control IgG) or G28-5 (an anti-CD40monoclonal antibody) or were preincubated on CD32-L cells and at thesame time treated with IL-4. The preincubation with the CD32-L cellstogether with IL-4 treatment have been shown to be an efficient form ofcross-linking the CD40 molecule other than direct gene transfection.

After three days of culture at 37° C., these cells were treated withmitomycin C to prevent their proliferation and then used to stimulateallogeneic T cells. Prior to this co-culture, the different aliquots ofCLL cells had either been treated with the anti-CD40 monoclonal antibodyor had been infected with the gene therapy vector containing either thelac-Z or murine CD40 ligand gene at a stimulator ratio of 1:10. Aftertwo days of culture at 37° C., interferon gamma (IFN_(γ)) production wasmeasured by ELISA assay. After five days of co-culture at 37° C., theincorporation of ³H-thymidine into replicating cells was measured afteran eight hour pulse label. The results of this assay are shown in TableII below and in FIG. 5.

In another experiment, CLL B cells infected with the gene therapy vectorcontaining the CD40 ligand gene were evaluated for their ability to actas stimulator cells in an allogeneic mixed lymphocyte T cell reaction(MLTR). In parallel, the stimulatory capacity of controllac-Z-vector-infected CLL cells and CLL B cells that had been culturedwith CD32-L cells and an anti-CD40 CD40 mAb (G28-5) or an isotypecontrol Ig, was also examined as described in Ranheim, E. A. and T. J.Kipps, J. Exp. Med., 177:925-935 (1993), Clark, E. A. and J. A.Ledbetter, Proc. Natl. Acad. Sci. USA, 83:4494-4498 (1986), andBanchereau, J. et al., Science 251:70-72 (1991). Effector T cells from anon-related donor were co-cultured with the CLL stimulator cells at aneffector to target ratio of 4:1. After 18 h culture at 37° C., over 30%of the allogeneic CD3⁺ cells were found to express theactivation-associated antigen CD69 when cultured with CD154-CLL cells(data not shown). In contrast, less than 4% of the T cells expressedCD69 when co-cultured with uninfected or Ad-lacZ-infected CLL cells(data not shown).

Two days after the initiation of the MLTR, the concentrations of IFN_(γ)in the culture supernatants were assayed by ELISA. The supernatants ofthe MLTR stimulated with CLL cells infected with the accessory moleculeligand CD40L (FIG. 12A, CD154-CLL) contained significantly higher levelsof IFN_(γ) (306±5 ng/ml, m±SE, n=3) than that of MLTR culturesstimulated with the anti-CD40 mAb (FIG. 12A, αCD40-CLL) (23±3 ng/ml)(P<0.05, Bonferroni t-test). The latter was not significantly differentfrom that of MLTR cultures stimulated with control Ad-lacZ-infected CLLcells (FIG. 12A, lacZ-CLL) (43±10 ng/ml) (P>10.1, Bonferroni t-test).The supernatants of effector cells alone, or of MLTR cultures stimulatedwith uninfected CLL cells (FIG. 12A, CLL) or control Ig treated CLLcells (FIG. 12A, MOPC-CLL), did not contain detectable amounts ofIFN_(γ) (<2 ng/ml). Similarly, none of the leukemia B cell populationsproduced detectable amounts of IFN_(γ) when cultured alone, withoutadded effector T cells (data not shown).

After 5 days, cell proliferation was assessed by incorporation of³H-thymidine. Cultures with isotype control IgG-treated (FIG. 12B,MOPC-CLL) or uninfected (FIG. 12B, CLL) stimulator cells did notincorporate more ³H-thymidine than cultures without addedleukemia-stimulator cells (FIG. 12B, None). Ad-lacZ-infected CLL B cells(FIG. 12B, lacZ-CLL) also were unable to stimulate allogeneic T cells toincorporate amounts of 3H-thymidine that were much greater than that ofcontrol cultures. In contrast, anti-CD40-stimulated leukemia cells orCD154-CLL cells each induced significant effector cell proliferation(FIG. 12B, αCD40-CLL or. CD154-CLL) (P<0.05, Bonferroni t-test).Moreover, the amount of 3H-thymidine incorporated by cultures stimulatedwith CD154-CLL cells (41,004±761 cpm (m±SE), n=3) was significantlygreater than that of cultures stimulated with equal numbers of αCD40-CLLcells (22,935±1,892 cpm; n=3) (P<0.05, Bonferroni t test). However,neither of these mitomycin-C-treated leukemia cell populationsincorporated ³H-thymidine when cultured without effector T cells (datanot shown). Also, as described for the MLTR between allogeneic T cellsand CD40-stimulated CLL cells {6549, 7167, 7168}, allogeneic T cellproliferation in response to CD154-CLL could be inhibited by CTLA-4-Igor CD11a mAb when added at the initiation of the MLTR, indicating thatrespective interactions between CD80/CD86 and CD28, or CD54 andCD11a/CD18, contribute to the noted allogeneic T cell reaction (data notshown). TABLE II Allogeneic T cell responses to CLL cells infected withmCD40-L adenovirus Allogeneic response (mean ± SEM) % positive cellsIFNγ Human 3H-TdR production Stimulators mCD40-L CD80 uptake (cpm)(ng/ml) None (t cells only) — — 3577 ± 821   n.d.* CLL with: Noactivation 0 1.4 4577 ± 1097 n.d. MOPC21 0 1.0 5259 ± 1788 n.d. G28-5 026.7 22935 ± 1892  22.3 ± 1.6 lac-Z adeno 0 4.8 9037 ± 1781  43.2 ± 10.5mCD40-L adeno 17.5 19.7 41004 ± 761  305.7 ± 4.5 *n.d. - not detectable

iii. Stimulation of Gamma Interferon by CLL Cells Containing anAccessory Molecule Ligand Gene

The function of CLL cells containing an accessory molecule ligand gene(mouse CD40 ligand) was analyzed by determining the ability of thosecells to activate T lymphocytes. The procedure was performed as follows:allogeneic T lymphocytes from a healthy donor (greater than 90% CD3⁺)were purified using magnetic beads and monoclonal antibodies specificfor the CD14 and CD19 antigen. These allogeneic T lymphocytes then werecultured together with MMC-treated CLL cells which were infected withthe accessory molecule ligand gene (murine CD40 ligand) or the lac-Zgene. This co-culture was performed in RPMI-1640 medium containing 10%fetal calf serum. After culture for 24 hours, the cells were collectedand analyzed to determine the expression of the antigen CD69 on the Tlymphocytes using a standard FACS sorting protocol. The cell culturesupernatants were collected after two days in culture and tested todetermine the concentration of human interferon gamma using an ELISAassay. A portion of the CLL cells containing an accessory moleculeligand gene (murine CD40 ligand) and a portion of the cells containingthe adenovirus expressing the lac-Z were cultured in the presence ofhuman interleukin 4 IL-4 (5 ng/mL). The production of interferon gammaby allogeneic T lymphocytes in the presence of this amount of humaninterleukin 4 was also analyzed. The results from these analyses areshown in FIG. 6.

As can be seen, the human CLL cells containing the accessory moleculeligand gene (murine CD40) produced substantially higher concentrationsof interferon gamma in the cell culture supernatant when compared to CLLcells which contained the lac-Z gene. The increased production ofinterferon gamma (IFN_(γ)) by T lymphocytes exposed to CLL cellscontaining the accessory molecule ligand gene indicates that these CLLcells containing the accessory molecule ligand genes were effective inproducing an enhanced immune response.

iv. Stimulation of Allogeneic T Cells Pre-Exposed to Non-Modified CLL BCells Containing an Accessory Molecule Ligand Gene

Prior studies indicated that antigen presentation to T cells, in theabsence of the signals derived from costimulatory molecules such asCD28, can lead to specific T cell clonal anergy. For this reason,allogeneic T cells that had previously been cultured, with non-modifiedCLL B cells lacking expression of CD80 and other immune accessorymolecules, were tested for their ability to respond to CLL cellscontaining the CD40 ligand gene. Allogeneic effector cells did notincorporate more ³H-thymidine in response to non-modified CLL cells(FIG. 12C, CLL), or control CLL cells infected with Ad-lacZ (FIG. 12C,lacZ-CLL), than when they were cultured alone (FIG. 12C, None). Incontrast, even after prior co-culture with non-modified CLL B cells,allogeneic effector cells could still be induced to proliferate (FIG.12C, CD154-CLL) or to produce IFN_(γ) (FIG. 12D, CD154 CLL) in responseto cells expressing an accessory molecule ligand. Although modestamounts of IFN_(γ) were detected in the supernatants of such secondarycultures when Ad-lacZ-infected leukemia cells were used as stimulatorcells (FIG. 12D, lacZ-CLL), this level was significantly lower than thatnoted for secondary cultures with Ad-CD40-ligand-infected CLL cells(FIG. 12D, CD154-CLL) (P<0.05, Bonferroni t-test). Similarly, thesupernatants of the leukemia cells alone (data not shown), and theeffector cells alone (FIG. 12D, None), of the MLTR cultures stimulatedwith uninfected CLL cells (FIG. 12D, CLL), contained negligible amountsof IFN_(γ) (<2 ng/ml). These results indicate that allogeneic effectorcells cultured with nonmodified CLL B cells are not precluded fromresponding to CLL B cells infected with a gene therapy vector containingthe accessory molecule ligand gene.

V. Autologous T Cell Responses to CLL Cells into which a Gene TherapyVector Encoding a Murine Accessory Molecule Ligand Gene has beenIntroduced

T cells isolated from the blood of CLL patients were examined for theirability to respond in vitro to autologous CLL B cells containing a genetherapy vector which encodes the murine accessory molecule, CD40 ligand.T cells were isolated to >95% purity, and then co-cultured withmitomycin-C-treated autologous leukemia cells in serum-free AIM-V mediumsupplemented with exogenous interleukin-2 at 25 U/ml. Modest³H-thymidine incorporation (≦10,000 cpm) was detected in cultureswithout added stimulator cells, secondary in part to the exogenous IL-2(FIG. 13A, and data not shown). The level of T cell proliferation,however, did not increase in response to uninfected CLL cells (FIG. 13A,CLL) or Ad-lacZ-infected CLL cells (FIG. 13A, lacZ-CLL). In contrast,CLL cells infected with a gene therapy vector containing the accessorymolecule ligand (FIG. 13A, CD154-CLL) induced autologous T cells toincorporate significantly more ³H-thymidine (17, 368±1,093 cpm, n=3)than any of the control cultures (P<0.05, Bonferroni t-test).Furthermore, the MLTR stimulated with CLL cells infected with a vectorencoding an accessory molecule ligand (CD40L) also generatedsignificantly more IFN_(γ) (165±3 ng/ml, n=3) than any of the othercultures (FIG. 13B)(P<0.05, Bonferroni t-test).

The T cells were harvested after 5 days from the autologous MLTR andassessed for CTL activity against autologous CLL B cells. T cellsco-cultured with autologous CD40-ligand-CLL cells developed CTL activityfor non-modified CLL B cells, effecting 40.1% lysis (±2.3%) at an E:Tratio of 2:1 (FIG. 13C, CD154). However, such T cells did not developdetectable CTL activity for the same target cells in the controlreactions, when co-cultured with uninfected or Ad-lacZ-infected CLLcells (FIG. 13C).

vi. Specificity of CTL Stimulated by Autologous CD40-Ligand-CLL B Cellsfor Allogeneic CLL B Cells

Effector cells stimulated with autologous CD40-ligand-CLL were evaluatedfor their ability to secrete IFN_(γ) or manifest CTL activity againstallogeneic CLL B cells (FIG. 14). After 5 days of autologous MLTR withCD154-CLL or lacZ-CLL, T cells were isolated by Ficoll density gradientcentrifugation, washed extensively, and then cultured in media for 24 h.Washed T cells were mixed with autologous (“Auto CLL”, solid bar) orallogeneic (“Allo-1 CLL” or “Allo-2 CLL”, shaded or hatched bars) targetCLL B cells. T cells stimulated in the autologous MLTR withCD40-ligand-CLL cells, but not with lacZ-CLL cells, producedsignificantly more IFN_(γ) in response to secondary culture withnon-modified autologous CLL B cells than with allogeneic CLL B cells(FIG. 14A) (P<0.05, Bonferroni t-test). Furthermore, T cells stimulatedwith CD40-ligand-CLL cells, but not with lacZ-CLL cells, were cytotoxicfor autologous CLL cells, but not allogeneic CLL cells (FIG. 14B).Similar results were obtained with the autologous MLTR-activated T cellsof the allogeneic donor, again demonstrating specific cytotoxicity forautologous CLL B cells (data not shown). Finally, W6/32, a mAb to classI major histocompatibility complex (MHC I) antigens could significantlyinhibit the cytotoxicity of T cells stimulated with CD40-ligand-CLLcells for autologous CLL B cells (FIG. 14C, αHLA-class I) ) (P<0.05,Bonferroni t-test). Such inhibition was not observed with mAb specificfor MHC class II antigen (FIG. 14C, αHLA-DP), mAb specific for theFas-ligand (FIG. 14C, αFasL), or an isotype control mAb of irrelevantspecificity (FIG. 14C, MOPC-21). Collectively, these studies indicatethat Ad-CD40-ligand-infected CLL cells can induce an autologousanti-leukemia cellular immune response in vitro, leading to thegeneration of MHC-class I-restricted CTL specific for autologousnon-modified leukemia B cells.

e. Transactivation of Non-Infected Bystander Leukemia B Cells byAd-CD40L CLL Cells

To address whether the changes in tumor marker expression (described insection 1di.) resulted from intracellular versus intercellularstimulation, the effect of culture density on the induced expression ofCD54 and CD80 following infection with adenovirus gene therapy vectorencoding the accessory molecule ligand (CD40L, or CD154) was examined.After infection, CLL cells were cultured at standard high density (e.g.1×10⁶ cells/ml) or low density (e.g. 2×10⁵ cells/ml) for 3 days at 37°C. Cells plated at high density contained homotypic aggregates, whereascells plated at low density remained evenly dispersed and withoutsubstantial cell-cell contact (data not shown). Despite expressingsimilar levels of heterologous CD154, CD154-CLL B cells cultured at highdensity were induced to express higher levels of CD54 and CD80 thanCD154-CLL cells cultured at low density (FIG. 15A). The stimulationachieved at high density could be inhibited by culturing the cells witha hamster anti-mouse CD154 mAb capable of blocking CD40<->CD154interactions (FIG. 15B, αCD154 Ab). Collectively, these studies indicatethat CD154-CLL cells can activate each other in trans and that surfaceexpression of CD154 is necessary for optimal leukemia cell stimulation.

In addition, Ad-CD154-infected, uninfected, Ad-lacZ-infected, orG28-5-stimulated CLL cells were labeled with a green-fluorescence dye toexamine whether CD154-CLL could stimulate non-infected bystanderleukemia cells. Dye-labeled cells were used as stimulator cells forequal numbers of non-labeled syngeneic CLL B cells. After 2 days'culture, stimulator cells cultured by themselves retained thegreen-fluorescence dye, allowing such cells to be distinguished fromnon-labeled CLL cells by flow cytometry. Bystander(green-fluorescence-negative) CD19⁺ CLL B cells were induced to expressCD54 (FIG. 15C, right histogram) or CD86 (FIG. 15D, right histogram)when co-cultured with Ad-CD154-infected leukemia B cells, but not withmock infected CLL cells (FIGS. 15C and 15D, left histograms),G28-5-stimulated CLL cells, or Ad-lacZ-infected CLL cells (data notshown). As expected, these bystander (green-fluorescence-negative) CLLcells also were negative for heterologous CD154.

f. Treatment of Leukemia with Gene Therapy Vectors Encoding an AccessoryMolecule Ligand

FIG. 24 shows an outline for a clinical trial for testing treatment of Bcell CLL with adenovirus gene therapy vectors encoding modified CD40ligand. Leukemia cells harvested by pheresis are infected withreplication-defective vectors that encode the modified CD40 ligand.Following expression of this protein, the cells will be administeredback to the patient for the purpose of stimulating a hostanti-leukemia-cell immune response. This strategy is far superior to onethat uses gene therapy to affect expression of only one immunestimulatory molecule on the leukemia cell surface. Indded, this strategyresults in the leukemia cells expressing an array of immune-stimulatoryaccessory molecules and cytokines, as well as a molecule that can affectthe same changes in leukemia cells of the patient that were neverharvested.

2. Expression of Chimeric Accessory Molecule Ligand Genes

The chimeric accessory molecule ligand genes described below areprepared using standard techniques as described herein.

a. Preparation of Chimeric Accessory Molecule Ligand Genes UtilizingDomains from Two Different Accessory Molecule Genes

The human CD40 ligand gene was isolated from RNA prepared from T cellswhich had been activated by an anti-CD3 monoclonal antibody using 5′ and3′ primers together with well known PCR methods. Chimeric accessorymolecule genes of human CD40 ligand and murine CD40 ligand areconstructed from the newly cloned human CD40 ligand gene and mouse CD40ligand gene described herein as SEQ ID NO: 2. The transmembrane andcytoplasmic domains of human CD40 ligand genes are exchanged with thoseof the murine CD40 ligand gene and designated H(Ex)-M(Tm-Cy) CD40ligand. These chimeric accessory molecule ligand genes are producedusing the gene conversion technique described as SOEN which has beenpreviously described by Horton, Mol. Biotechnol., 3:93 (1995). A diagramdepicting the chimeric accessory molecule ligand genes which areproduced is shown in FIG. 4. The nucleotide sequences of each of theserespective chimeric accessory molecule ligand genes is designated SEQ IDNOS: 3-7 as indicated in the Table below. TABLE III Chimeric AccessoryMolecule Ligand Gene SEQ ID NO: HuIC/HuTM/MuEX CD40-Ligand SEQ ID NO: 3HuIC/MuTM/HuEX CD40-Ligand SEQ ID NO: 4 HuIC/MuTM/MuEX CD40-Ligand SEQID NO: 5 MuIC/HuTM/HuEX CD40-Ligand SEQ ID NO: 6 MuIC/MuTM/HuEXCD40-Ligand SEQ ID NO: 7

Adenovirus vectors encoding each of the chimeric accessory moleculesshown in FIG. 2 are constructed using the methods described inExample 1. Each of these constructs are then transfected into eitherHeLa cells or CLL cells according to the methods of Example 1.

b. Expression of Chimeric Accessory Molecule Ligands on CLL and HeLaCells

The expression of each of the chimeric accessory molecule ligand genesconstructed above is analyzed by using FACS analysis as specified inExample 1. The appropriate monoclonal antibody immunospecific for theexternal domain of either human or mouse CD40 ligand is selected andused to determine the level of expression of the chimeric accessorymolecules on the surface of these cells. After appropriate analysis andpreparation of appropriate histograms, the expression of chimericaccessory molecules containing at least a portion of the murine CD40ligand gene is confirmed.

c. Function of Chimeric Accessory Molecule Ligands

CLL cells are infected with various MOI of the mCD40L adenovirus andthen cultured in 48 or 24 well tissue culture plates for various timesafter infection (48, 72, and 96 hours). The CD19⁺ B cells are thenanalyzed by multiparameter FACS analysis for induction of CD80 and CD54expression using fluroescein isothiocyanate-conjugated mAb specific foreach respective surface antigen as described in Example 1. Increasedamounts of CD54 and CD80 are found on cells which have the chimericaccessory molecules containing the domain or domains derived from themouse CD40 ligand gene.

Further analysis of the cells containing the chimeric accessory moleculegenes is carried out according to Example 1(d). The cells containing thechimeric accessory molecule genes which contain the domains derived fromthe murine CD40 ligand gene are able to stimulate the production ofgamma interferon and T cell proliferation.

d. Expression of Chimeric Accessory Molecule Genes which ContainProximal Extracellular Domains from Two Different Accessory Moleculesfrom the Same Species

A chimeric accessory molecule ligand gene is prepared which contains theproximal extracellular domain from the human CD70 gene (Domain III) withthe remainder of the domains derived from the human CD40 ligand gene.This gene is prepared using standard biologic techniques as previouslydescribed herein. This chimeric accessory molecule ligand gene has theDNA sequence shown as SEQ ID NO: 19. A different chimeric accessorymolecule ligand gene is prepared which contains the proximalextracellular domain from the murine CD40 ligand gene with the remainderof the domains derived from the human CD40 ligand gene. This gene isprepared using standard techniques as previously described herein. Thischimeric accessory molecule ligand gene has the DNA sequence shown asSEQ ID NO: 20.

The chimeric accessory molecule genes shown as SEQ ID NOS: 19 and 20 areinserted into the appropriate vectors as described in Example 1 andintroduced into human neoplastic cells. The expression of that chimericaccessory molecule gene in the cells is determined as was described inExample 1.

The chimeric accessory molecule encoded by each of these chimericaccessory molecule genes is found on the surface of the human neoplasticcells using the FACS analysis described in Example 1. Increased amountsof CD54 and CD80 are found on the cells containing the chimericaccessory molecule genes using the techniques described in Example 1.The cells containing the chimeric accessory molecule gene are able tostimulate the production of gamma interferon and T cell proliferation asdescribed and assayed according to Example 1.

3. Augmentation of Vaccination Using Vectors Encoding AccessoryMolecules

The following procedures were used to demonstrate the augmentation of avaccination protocol using a gene therapy vector encoding an accessorymolecule.

a. Augmentation of the Antibody Response in Mice Co-Injected with anAccessory Molecule Gene Therapy Vector and placZ

Three different gene therapy constructs were prepared using standardtechniques including those techniques described herein. The first was acontrol gene therapy vector, pcDNA3, which did not contain any gene. Thesecond, placZ, contained the Lac-Z gene which encoded β-galactosidase(β-gal). The third, p-mCD40L, contained the murine CD40 ligand genedescribed in Example 1.

Prior to any immunizations, serum was isolated from 6-8 week oldBALB/c-mice to determine the amount of any initial antibodies toβ-galactosidase. Each animal was injected i.m. with 100 micrograms ofplasmid DNA per injection. Four separate injections were given at oneweek intervals.

Prior to the third injection, the animals were bled to monitor the earlyantibody response to β-gal. One week after the final injection ofplasmid DNA, the animals were bled to monitor the late antibody responseto beta-galactosidase. To test the sensitivity of the assay, knownamounts of anti-β-gal antibodies isolated from an anti-β-gal antiserumwere tested in parallel.

Serum dilutions of 1:40, 1:200, or 1:1000 were tested in an ELISA foranti-β-gal antibodies. For this, polystyrene microtiter ELISA plateswere coated with β-gal at 10 microgram/ml in phosphate buffered saline.The plates were washed thrice with blocking buffer containing 1% bovineserum albumin (BSA), 0.2% Tween 20 in borate buffered saline (BBS) (0.1Mborate, 0.2M NaCl, pH 8.2). 50 microliters of diluted serum were addedto separate wells. After at least 1 hour at room temperature, the plateswere washed thrice with blocking buffer and then allowed to react withalkaline phosphatase-conjugated goat anti-mouse IgG antibody. One hourlater, the plates again were washed four times with blocking buffer andincubated with 25 ml of TMB peroxidase substrate (Kirkegaard & Perry,Gaithersburg, Md.). The absorbance at 405 nm of each well was measuredusing a microplate reader (Molecular devices, Menlo Park, Calif.). Thehigher the O.D. reading, the greater the amount of specific antibody inthe sample.

The data for each of two experiments are provided in Tables IV and Vwhich follow on separate sheets. The results are summarized in Tables VIand VII collating the data from the two experiments is provided as well.On the summary page n stands for the number of animals in each of thefour groups. S.D. stands for standard deviation and Avg. is the averageO.D. reading for all the animals in a particular group.

The results of Group 4 demonstrate that the use of a gene therapy vectorencoding an accessory molecule ligand (CD40L) enhances the immunizationagainst β-gal encoded by a genetic or gene therapy vector. The averageO.D. reading of the 1:40 dilution of the sera from animals of this groupis significantly higher than that of groups 1, 2, and 3 (P<0.05,Bonferroni t tests, see Table VII).

Data from an additional experiment further reinforce the finding thatthe gene therapy vector encoding an accessory molecule ligand enhancesimmunization against β-gal (FIG. 16). Here, pCD40L and placZ wereco-injected into skeletal muscle, to test for enhancement of the immuneresponse to placZ, a pcDNA3-based vector encoding E. coliβ-galactosidase. The relative anti-β-gal Ab activities were determinedvia ELISA. As expected, mice injected with either the non-modifiedpcDNA3 vector or pCD40L alone did not produce detectable antibodies toβ-gal (FIG. 16A). Mice were injected with either 100 μg pcDNA3(checkered bar), 50 μg pcDNA3+50 μg pCD40L (lined bar), 50 μg pcDNA3+50μg placZ (striped bar), or 50 μg pCD40L+50 μg placZ (solid bar). On theother hand, mice that received placZ and pcDNA3 developed detectableanti-β-gal antibodies one week after the fourth and final injection, atd28. Mice that received placZ and pCD40L developed higher titers ofanti-β-gal antibodies than mice injected with placZ and pcDNA3. FIG.16B, ELISA analyses of serial dilutions of sera collected at d28, showsthat mice co-injected with placZ and pCD40L had an eight-fold highermean titer of anti-β-gal antibodies at d28 than mice treated withplacZ+pcDNA3.

i. Immunoglobulin Subclass Production Stimulated by Accessory MoleculeVector Co-Injection

Despite enhancing the titer of the anti-β-gal antibody response, thesubclass of anti-β-gal IgG induced by injection of placZ was not alteredby the co-injection of pCD40L. IgG_(2a) anti-β-gal antibodiespredominated over IgG₁ subclass antibodies in the sera of mice injectedwith either placZ and pcDNA3 or placZ and pCD40L (FIG. 17). Alsodepicted are the ELISA O.D. measurements of anti-β-gal IgG₁ andanti-β-gal IgG₂a present in the pre-immute sera (striped bar) orpost-immune sera (solid bar), collected at d28) of each group of mice,injected as indicated on the abscissa. In contrast, BALB/c mice injectedwith β-gal protein developed predominantly IgG₁ anti-β-gal antibodies,and no detectable IgG_(2a) anti-β-gal antibodies.

ii. Augmentation of Vaccination by Accessory Molecule Vector RequiresCo-Injection with placZ at the Same Site

The adjuvant effect of the pCD40L plasmid on the anti-β-gal antibodyresponse was noted only when it was injected into the same site as placZ(FIG. 18). Groups of BALB/c mice (n=4) received intramuscular injectionsof placZ and pCD40L together at the same site, or as simultaneousseparate injections at distal sites (right and left hind legquadriceps). A control group received intramuscular injections of placZand pcDNA3 at the same site. Animals were bled at d28 and the seratested for anti-β-gal Ab at different dilutions, as indicated on theabscissa. The graph illustrates a representative experiment depictingthe mean O.D. at 405 nm of replicate wells of each of the serum samplesfor each group, at a 1:40, 1:200, or 1:1000 dilution. Animals injectedsimultaneously with placZ and pCD40L, but at different sites, did notdevelop detectable anti-β-gal antibodies until d28. Moreover, theanti-β-gal antibody titers of the sera from such animals at d28 weresimilar to that of mice that received placZ and pcDNA3, andsignificantly less than that of animals that received placZ and pCD40Ltogether at the same site.

iii. Augmentation of Vaccination when Accessory Molecule Vector andplacZ are Co-Injected into Dermis

The pCD40L plasmid also enhanced the anti-β-gal antibody response toplacZ when injected into the dermis. In the experiment shown in FIG. 19,mice received intradermal injections, near the base of the tail, witheither 50 μg pcDNA3 (checkered bar), 25 μg pcDNA3+25 μg pCD40L (linedbar), 25 μg pcDNA3+25 μg placZ (striped bar), or 25 μg pCD40L+25 μgplacZ (solid bar). Injections, bleeds and ELISA analyses were performedas in FIG. 16A. The checkered bar and lined bar groups each consisted of8 mice while the striped bar and solid bar groups each consisted of 12mice. The height of each bar represents the mean O.D. of sera at a 1:40dilution of each group±S.E. A statistical analysis of the data indicatedthat the striped bar and solid bar groups are independent (P<0.05). Asobserved with intramuscular injection, mice co-injected with placZ andpCD40L developed detectable serum anti-β-gal antibodies one weekfollowing the second injection (d14), and two weeks earlier than miceinjected with placZ and pcDNA3. Moreover, these animals also had aneight-fold higher mean titer of anti-β-gal antibodies than mice of theplacZ-injected group at d28. Mice injected with either the non-modifiedpcDNA3 vector or pCD40L alone did not produce detectable antibodies toβ-gal.

b. Augmentation of the CTL Response in Mice Co-Injected with anAccessory Molecule Gene Therapy Vector and placZ

The ability of pCD40L to enhance induction, by placZ, of CTL specificfor syngeneic β-gal-expressing target cells was tested. BALB/c miceco-injected with pCD40L and placZ into skeletal muscle (FIG. 20A) ordermis (FIG. 20B) generated greater numbers of CTL specific for P13.2, aplacZ transfected P815 cell line, than mice co-injected with placZ andpcDNA3. At a 5:1 effector:target ratio, the splenocyte effector cellsfrom mice that received intramuscular injections of placZ and pCD40Lachieved greater than 20% specific lysis of P13.2. In contrast, whensplenocytes of mice that received the control injection with placZ andpcDNA3 were used, a 9-fold greater ratio of effector to target cells wasrequired to achieve this level of specific lysis. Similarly, thesplenocyte effector cells from mice that received intradermal injectionsof placZ and pCD40L killed more than 50% of the P13.2 cells ateffector:target ratios of 4:1. To achieve comparable levels of specificlysis required eight-fold higher effector:target ratios usingsplenocytes from mice that received intradermal injections of placZ andpcDNA3. Nevertheless, the splenocytes of mice co-injected with pCD40Land placZ did not have greater non-specific CTL activity for P815 cellsthan that of mice that received placZ along with pcDNA3 (FIG. 20). Asexpected, the splenocytes from mice that received injections of pcDNA3alone, or pcDNA3 and pCD40L, did not mediate specific lysis of P13.2 orP815 cells. TABLE IV Experiment #1 Injections of plasmid DNA i.m.: Apr.3,1996; Apr. 10,1996; Apr. 17,1996; Apr. 24,1996 ELISA for anti-betagalactosidase Dilution of Pre-Bleed (4/3) Dilution of Bleed (4/17

antibodies: Animal 1/140 1/200 1/1000 1/140 1/200 1/1

Group 1 0.09 0.11 0.09 0.06 0.06

pcDNA3 (p-control, 100 mcg) 2 0.11 0.09 0.09 0.07 0.07

(Control vector) 3 0.12 0.11 0.10 0.09 0.09

4 0.11 0.10 0.10 0.08 0.11

Avg. 0.11 0.11 0.11 0.11 0.11

S.D. 0.01 0.01 0.01 0.01 0.02

p-lacZ (50 mcg) + 5 0.13 0.10 0.10 0.07 0.11

p-Control (50 mcg) 6 0.10 0.11 0.10 0.07 0.06

7 0.19 0.10 0.18 0.07 0.07

8 0.10 0.09 0.10 0.08 0.07

Avg. 0.13 0.10 0.12 0.07 0.08

S.D. 0.04 0.01 0.04 0.01 0.02

p-lacZ (50 mcg) + 27  0.06 0.06 0.06 0.13 0.11

pRcCMV-mCD40L (p-mCD40L, 50 mcg) 18  0.06 0.06 0.06 0.27 0.13

19  0.06 0.06 0.06 0.23 0.19

20  0.06 0.06 0.06 0.23 0.19

Avg. 0.06 0.06 0.06 0.74 0.47

S.D. 0.00 0.00 0.00 1.06 0.66

TABLE V Experiment #2 Injections of plasmid DNA i.m.: Jun. 5,1996; Jun.12,1996; Jun. 19,1996; Jun. 26,1996 Dilutions of sera for anti-betaDilution of Pre-Bleed (6/5) Dilution of Bleed (7/1

galactosidase antibodies: Animal 1/140 1/200 1/1000 1/140 1/200 1/1

Group  9 0.02 0.02 0.06 0.04 0.01

p-Control (50 mcg) + 10 0.06 0.02 0.10 0.02 0.02

p-mCD40L (50 mcg) 11 0.02 0.02 0.07 0.03 0.01

12 0.06 0.03 0.05 0.16 0.04

Avg. 0.04 0.04 0.04 0.04 0.04

S.D. 0.02 0.01 0.02 0.07 0.01

p-lacZ (50 mcg) +  5 0.02 0.03 0.02 0.06 0.04

p-Control (50 mcg)  6 0.03 0.02 0.03 0.14 0.03

 7 0.56 0.13 0.06 0.29 0.06

 8 0.01 0.02 0.05 0.06 0.02

Avg. 0.15 0.05 0.04 0.13 0.04

S.D. 0.27 0.05 0.02 0.11 0.02

p-lacZ (50 mcg) + 13 0.23 0.06 0.05 0.28 0.07

p-mCD40L (50 mcg) 14 0.02 0.02 0.03 0.04 0.02

15 0.02 0.02 0.02 0.89 0.21

16 0.05 0.04 0.02 0.11 0.04

Avg. 0.08 0.04 0.03 0.33 0.08

S.D. 0.10 0.02 0.02 0.39 0.09

TABLE VI Pre-Immune @ Early @ beta-gal beta-ga

Summary 1/140 1/200 1/1000 1/140 1/200 1/1

1) p-Control Avg. 0.11 0.11 0.11 0.11 0.11

(n = 4) S.D. 0.01 0.01 0.01 0.01 0.02

2) p-mCD40L + Avg. 0.04 0.04 0.04 0.04 0.04

p-Control (n = 4) S.D. 0.02 0.01 0.02 0.07 0.01

3) p-lacZ + Avg. 0.11 0.04 0.04 0.11 0.03

p-Control (n = 8) S.D. 0.22 0.04 0.01 0.09 0.02

4) p-lacZ + Avg. 0.11 0.04 0.03 0.25 0.06

p-mCD40L S.D. 0.10 0.02 0.01 0.32 0.07

(n = 8) Anti-beta- galactosidase standard: 67 ng 22 ng 7.4 ng 2.5 ng .82ng .27 ng O.D. 3.01 2.98 2.05 1.10 0.52 0.26 3.14 3.14 2.25 1.20 0.560.26

TABLE VII BONFERRONI t-TESTS Comparison Difference of means t P < .05 4vs 2: 2.06 − 0.04 = 2.02 3.782 Yes 4 vs 1: 2.06 − 0.11 = 1.95 3.651 Yes4 vs 3: 2.06 − 0.61 = 1.45 3.325 Yes 3 vs 2: 0.61 − 0.04 = 0.57 1.067 No3 vs 1: 0.61 − 0.11 = 0.50 Do not test 1 vs 2: 0.11 − 0.04 = 0.07 Do nottest Degrees of freedom: 20 ONE WAY ANALYSIS OF VARIANCE Group N MeanStd Dev SEM 1 4 0.11 0.01 0.00 2 4 0.04 0.04 0.02 3 8 0.61 1.11 0.39 4 82.06 0.97 0.34 5 4 1.51 0.77 0.38 6 4 1.14 0.53 0.26 7 4 0.83 0.43 0.22ONE WAY ANALYSIS OF VARIANCE Source of Variation SS DF Variance Est (MS)Between Groups 18.29  6 3.05 Within Groups 18.39 29 0.63 Total 36.69 35$F = {\frac{s2\_ bet}{s2\_ wit} = {\frac{MSbet}{Mswit} = {\frac{3.05}{0.63} = {{4.81\quad P} = 0.0002}}}}$

4. Treatment of Neoplasia Using a Gene Therapy Vector Containing anAccessory Molecule Gene or Chimeric Accessory Molecule Gene a. Treatmentof Neoplasia in Mice

The treatment of a neoplasia in a mouse model system has beendemonstrated using the genes encoding accessory molecule ligands of thepresent invention. Gene therapy vectors containing an accessory moleculeligand gene (murine CD40 ligand) were prepared as has been previouslydescribed in the above examples. These gene therapy vectors were used tointroduce that accessory molecule ligand gene into neoplastic cells,Line1 cells, from a tumor which originated in BALB/c mice. The accessorymolecules were introduced into the neoplastic cells according to theabove examples. The expression of the accessory molecule ligand on thesurface of these neoplastic cells was confirmed using flow cytometry ashas been described in the above examples.

The effectiveness of the accessory molecule ligand genes for treatingneoplasia was shown as follows. Female BALB/c mice (6-8 weeks old) wereinjected i.p. with 1.0×10⁵ irradiated Line1 neoplastic cells. Theneoplastic Line1 cells are derived from a spontaneous lungadenocarcinoma in a BALB/c mouse. This neoplastic cell has beendescribed by Blieden et al., Int. J. Cancer Supp., 6:82 (1991). Otherfemale BALB/c mice were injected i.p. with 1.0×10⁵ irradiated Line1tumor cells that had previously been transduced with the gene therapyvector encoding the accessory molecule ligand gene (murine CD40) asdescribed above.

Each group of mice was allowed to generate an immune response for 10days. After 10 days each mouse was challenged with 1.0×10⁴ live,non-irradiated Line1 neoplastic cells. These mice were then monitoredfor the formation of tumors and then sacrificed when the tumors grew to2.0 cm because of morbidity. The results of this monitoring are shown inFIG. 7. As can be seen by FIG. 7, the mice immunized with the neoplasticcell expressing the accessory molecule ligands of the present inventionon the cell surface remained free of tumor throughout the experiment.Mice immunized with the neoplastic cells not having the accessorymolecule ligand genes of the present invention succumbed to tumor 50days after challenge with the neoplastic cells.

FIG. 21 demonstrates downmodulation of human CD40L, but not murineCD40L, in lung tumor cell lines that express CD40. Human cell lines HeLa(CD40-negative cervical carcinoma, FIG. 21A), A427 (CD40-negative lungcarcinoma, FIG. 21B), NCI 460 (weakly CD40-positive lung large cellcarcinoma, FIG. 21C), and SK-Mes-1 (strongly CD40-positive lung squamouscell tumor, FIG. 21D) were infected with adenovirus encoding lac-Z(Ad-LacZ), murine CD40L (Ad-mCD40L), and human CD40L (Ad-hCD40L) at anMOI of 0 (Blank), 1, and 10. 48 hours after infection, murine CD40L andhuman CD40L surface expression was determined. The percentage of cellsthat express ligand are plotted on the Y-axis. Human and mouse CD40L areexpressed at equal levels in CD40-negative cell lines. However, onlymurine CD40L expression is stable on cell lines that express CD40. Incontrast to mCD40L, human CD40L is downmodulated on CD40-positivetumors.

The data graphed in FIG. 22A show that CD40 binding induces expressionof tumor surface markers. Treating CD40-expressing lung cancer celllines with αCD40 mAb resulted in enhanced expression of the tumor cellsurface markers CD95 (Fas), CD54 (ICAM-1) and class I majorhistocompatability antigens (MHC I). NCI 460, a weakly CD40-positivelung large cell carcinoma, was incubated with a CD40-specific monoclonalantibody (thick line), or MOPC21, an isotype control mAb (thin line), onCD32-expressing mouse fibroblasts for 48 hours. Following the 48 hrincubation, the lung tumor cells were analyzed for CD95, CD54, and MHC-Iexpression by FACS.

FIG. 22B again shows downmodulation of human CD40L by CD40-positivetumor cells. HeLa (CD40-negative), CLL (CD40-positive), and SK-MES-1(CD40-positive) tumor cells were cocultured for 24 hours withCD3-activated normal donor T cells at a tumor cell:T cell ration of2.5:1. Following coculture, CD2-expressing T cells were analyzed forCD40L surface expression by FACS. Thin lines represent T cells stainedwith FITC-labeled isotype control antibody (MOPC21) and thick linesrepresent activated T cells stained with FITC-labeled αCD40L antibody(αCD154 antibody). The CD40-positive tumor cell lines, SK-MES-1, andCLL, do not express CD40 ligand on their surfaces.

5. Expression of the Human and Mouse Accessory Molecule Ligand, FasLigand, in Human Blood Lymphocytes a. Construction of a GeneticConstruct and Gene Therapy Vector Containing the Human and Mouse FasLigand Gene

Either the human accessory molecule ligand gene (human Fas ligand) orthe murine accessory molecule ligand gene (murine Fas ligand) wasconstructed utilizing the respective human and murine genes. An alteredaccessory cell molecule, in which a putative MMP-cleavage site wasremoved, was made and designated ΔFasL-pcDNA3. The nucleotide sequenceof ΔFasL-pcDNA3 is listed as SEQ ID NO: 40. Human Fas ligand nucleotides325 to 342, encoding six amino acids, are missing from ΔFasL. The designof ΔFasL was based on reasoning that Domain III contains sites mostaccessible to MMPs, and could thus be the target on the molecule forcleavage from the surface of the cell. Sequences of the human Fas ligandgene have been determined and are listed as SEQ ID NOS: 13 and 30(Genbank accession U11821). Sequences of mouse Fas ligand genes havebeen determined and are listed as SEQ ID NOS: 14 (C57BL/6, Genbankaccession U10984) and 31 (Balb/c, Genbank accession U58995). Thesequence of the rat Fas ligand gene has been determined and is listed asSEQ ID NO: 25 (Genbank accession U03470). Chimeric constructs are made,as described in Example 2 for CD40 ligand chimeric constructs, in whichDomain III of human Fas ligand is replaced with Domains of otherproteins, particularly proteins of the TNF family. Chimeric constructsinclude, but are not limited to, human Fas ligand with Domain IIIreplaced by Domain III of murine Fas ligand (chimeric sequence listed asSEQ ID NO: 37, sequence line-up shown in FIG. 37), or replaced by DomainIII of human CD70 (chimeric sequence listed as SEQ ID NO: 38, sequenceline-up shown in FIG. 38), or replaced with Domain I of human CD70(chimeric sequence listed as SEQ ID NO: 39, sequence line-up shown inFIG. 39). Chimeric constructs in which multiple domains, for example,two copies of human CD70 Domain III, are inserted into human Fas ligandin place of Domain III, are also made using methods described in ExampleI. Chimeric constructs in which synthetic sequences are used to replaceDomain III of human Fas ligand are also made.

1. Human Fas Ligand Cloning

The cDNA encoding human Fas-ligand was subcloned in the eukaryoticexpression vector pcDNA3. Normal donor blood lymphocytes were activatedfor 4 hours with 1 ng/ml PMA plus 0.5 uM ionomycin. Total RNA wasisolated with the Qiagen Rneasy kit. cDNA was then synthesized frompoly-A RNA with oligo-dT primers using the Gibco-BRL Superscript cDNAsynthesis kit. The gene encoding human Fas-ligand was then PCR amplifiedwith the Fas-ligand-specific primers (sense primer, SEQ ID NO: 32,antisense primer, SEQ ID NO: 33). The Fas-ligand PCR product was thensubcloned into pcDNA3 using standard molecular biology techniques.RT-PCR products, subcloned into pcDNA3, are designated hFasL-pcDNA3.

ii. Murine Fas Ligand Cloning

The murine Fas-ligand geneS from Balb/c and C57/BL6 strains of mice werealso amplified following activation of mouse splenocytes with PMA plusionomycin as described above, and amplified from poly-A synthesized cDNAas described above (sense primer, SEQ ID NO: 34, antisense primer, SEQID NO: 35). These genes were subcloned in the pTARGET expression vector(Promega, Madison, Wis.). RT-PCR products, subcloned into pcDNA3, aredesignated mFasL-pcDNA3.

iii. Adenovirus Vector Construction

For construction of adenovirus vectors encoding human Fas-ligand, murineFas-ligand or ΔFas-ligand, the cloned cDNA insert is subcloned into theplasmid pRc/RSV (Invitrogen, San Diego, Calif.) at the HindIII-Xbalsite. A BglII-Xhol fragment with the RSV promoter-enhancer and thebovine growth hormone poly-A signal sequence was subcloned into theBamHI-Xhol site of plasmid MCS(SK)pXCX2. The plasmid MCS(SK)pXCX2 is amodification of the plasmid pXCX2, in which the pBluescript polylinkersequence was cloned into the El region. The resulting plasmid then isco-transfected along with pJM17 into 293 cells using the calciumphosphate method. Isolated plaques of adenovirus vectors are picked andexpanded by infecting 293 cells. High titer adenovirus preparations areobtained, as described above which uses a cesium chloride gradient forconcentrating virus particles via a step gradient, with the densities of1.45 g/cm³ and 1.20 g/cm³, in which samples are centrifuged for 2 hoursin an SW41 rotor (Beckman, Brea, Calif.) at 25,000 rpm at 4° C. Thevirus band is desalted using a Sephadex G-25 DNA grade column(Pharmacia, Piscataway, N.J.), and the isolated virus is stored at −70°C.in phosphate-buffered saline with 10% glycerol. The titer of the virusis determined by infecting permissive 293 cells at various dilutions andcounting the number of plaques. Titers typically range from 10¹⁰ to 10¹²plaque forming units/ml. The adenovirus constructs are designatedAd-hFasL, Ad-mFasL and Ad-ΔFasL.

b. Introduction of the Murine and Human Fas Ligand Genes into HumanCells

The constructs hFasL-pcDNA3, mFasL-pcDNA3 and ΔFasL-pcDNA3 aretransfected into 293 via electroporation. The transfected cells areselected in medium containing G418. Fas-ligand transfectants arescreened for expression of the transgene using anti-Fas-ligand antibodyand flow cytometry. The methods used are similar to those described fortransfection of CD40L into CLL cells.

For FasL-adenovirus infection, 10⁶ freshly thawed and washed CLL cellsor HeLa cells are suspended in 0.5 to 1 mL of culture medium for cultureat 37° C. in a 5% CO₂-in-air incubator. Adenovirus are added to thecells at varying multiplicity of infection (MOI), and the infected cellsare cultured for 48 hours, unless otherwise stated, before beinganalyzed for transgene expression.

c. Expression of the Fas Ligand Genes in Human Cells

Mice with the lymphoproliferative or generalized lymphoproliferativedisorder are unable to delete activated self-reactive cells outside ofthe thymus. This is related to the fact that, in these mice,interactions between the Fas receptor and an accessory molecule ligand,Fas ligand, are defective. These animals develop numerous disordersincluding lymphadenopathy, splenomegaly, nephritis, and systemicautoimmune pathology which resembles that seen in patients with systemiclupus erythematosus or rheumatoid arthritis (RA). It is conceivable thatthe normal interactions between the Fas receptor and the accessorymolecule ligand that are responsible for clearance of activatedlymphocytes from joints may be impaired in RA patients.

RA synovial lymphocytes express the Fas receptor at a higher proportionthan that of matched RA blood lymphocytes to matched normal donor bloodlymphocytes. On the other hand, RA synovial lymphocytes express littleor no accessory molecule ligand. Since the RA synovial lymphocytes aresensitive to Fas-induced apoptosis, it is feasible that local expressionof Fas ligand in the RA joint could serve to eliminate the synovialmononuclear cells that potentially mediate RA autoimmune pathology.

FIG. 23 shows that Fas-ligand expression in lymphocytes is inhibited byexposure to RA synovial fluid. Normal donor blood T cells were activatedfor 5 hours with 1 ng/ml PMA plus 0.5 μM ionomycin. Cells were incubatedin the presence of rheumatoid arthritis blood plasma (circles), RAsynovial fluid (diamonds), or neither (squares). In addition, cells wereincubated with increasing concentrations of the MMP inhibitor BB94.Following activation, cells were analyzed for Fas-ligand surfaceexpression by FACS. The percentage of cells expressing Fas ligand areplotted in FIG. 23. This experiment demonstrates that there is afactor(s) present in RA synovial fluid and serum that prevents surfaceexpression of Fas-ligand.

d. Function of Human, Murine and Chimeric Accessory Molecule Ligand, FasLigand

To determine the capacity of the AFasL constructs, the above-mentionedtransfected cells are mixed with the Fas-ligand sensitive human T cellline, JURKAT. Following 4 hours coculture, the nonadherant JURKAT cellsare collected and evaluated for apoptosis. The fluorescent compound 3,31dihexyloxacarbocyanine iodide (DiOC₆) is used to evaluate for apoptosisusing a modification of a previously described protocol. For this, thecells are washed once at room temperature in phosphate buffered saline(PBS, pH 7.2). Cells are placed into separate wells of a 96 wellU-bottom plastic microtiter plate at 10⁵−5×10⁵ cells/well in 50 ml totalvolume. If indicated, saturating amounts of PE-conjugated antibodies areadded followed by addition of DiOC₆ and propidium iodide (PI). DiOC₆ andPI are used at 40 nM and 10 ng/ml final concentrations, respectively.The cells are then incubated 15 minutes in a 37° C., 5% CO₂ tissueculture incubator. The stained cells are then washed twice in ice coldPBS and ultimately suspended in 200 ml SM and analyzed by FACS. Deadcells and debris with characteristic forward and light scatter profilesand PI staining are excluded from analysis.

The ability of cells expressing ΔFasL-pcDNA3 to direct Fas-mediatedapoptosis of cells expressing CD95 is compared with that of cellsexpressing FasL-pcDNA3. Relative stability of the protein productsencoded by ΔFasL-pcDNA3 or FasL-pcDNA3 pre- and post-culture with RAsynovial fluid, and with or without the metalloproteinase inhibitors,are assessed via flow cytometry of cells expressing either ligand.

6. Treatment of Arthritis with Gene Therapy Vectors Encoding anAccessory Molecule Ligand, Fas Ligand

The heterologous Fas-ligand constructs, made as described above, thatshow the highest stability of expression in combination with thegreatest ability to mediate Fas-induced apoptosis, are used in genetherapy for RA. Potential therapeutic constructs are tested inwell-characterized mouse models of arthritis to assess efficacy andfunction in vivo.

a. Gene Therapy Treatment of Arthritis in Mice i. Mouse Models forArthritis

One mouse arthritis model is collagen-induced arthritis. It is knownthat injecting DBA/1 mice with type II collagen in complete Freund'sadjuvant (CFA) induces an arthritis with synovitis and erosions thathistologically resemble RA. For our studies, male DBA/I mice areimmunized with bovine type II collagen in complete Freund's adjuvant onday 0 and boosted itraperitoneally (i.p.) on day 21. On day 28, animalsare given an additional i.p. injection with lipopolysaccharide (LPS)and/or the same type collagen, or an injection of acetic acid alone.Swelling and/or redness of a fore or hind paw in animals immunized withcollagen typically is detected the third or fourth week following thesecond injection. The vertebrae are only rarely affected, and then onlyweeks after the initial peripheral joint swelling. Affected jointsdisplay initial histologic changes of synovial edema, followed bysynovial hyperplasia.

Another animal model, recently described by Kouskoff, V. et al., in Cell87:811-822 (1997) was generated fortuitously, by crossing a T cellreceptor (TCR) transgenic mouse line with the non-obese-diabetic (NOD)strain to produce the KRN×NOD mouse model of RA. The offspring of such amating universally develop a joint disease that is highly similar tothat of patients with RA. Moreover, the disease in these animals has anearly and reproducible time of onset and a highly reproducible course.The arthritis apparently is induced by chance recognition of anNOD-derived major histocompatibility complex (MHC) class II molecule bythe transgenic TCR, leading to breakdown in the general mechanisms ofself-tolerance and systemic self-reactivity.

ii. Relief of Arthritis Symptoms in Mice Treated with a Gene TherapyVector Encoding an Accessory Molecule Ligand

We have adapted and modified a protocol originally described by Sawchukand colleagues for micro-injecting adenovirus vectors into mouse joints.Using this procedure we can reproducibly inject a 5 μl volume into thearticular space of the mouse knee. In this procedure, the mice areanesthetized with metofane. A small incision of approximately 2-3 mm ismade with a #11 scalpel blade in the skin over the lateral aspect of theknee to visualize the patello-tibial ligament. We can inject up to 5 μlof fluid using a micro-100 μl-Hamilton syringe and a 30-gauge needle.After the injection, the knee incision is closed with Nexabond(Veterinary Products Laboratory). Our adenovirus titers typically exceed10¹⁰ plaque forming units (pfu) per ml, making it possible to deliver atleast 5×10⁸ pfu of virus in 5 ml into the knee joints, as outlinedabove. Control animals are injected with control Ad-lacZ vector, areplication-defective adenovirus vector lacking a transgene, or with thebuffer used to suspend the virus (10 mM Tris, 1 mM MgCl₂, 10% glycerol).

In another method, splenocytes will be harvested from mice that aresyngeneic to the host animal intended for adoptive transfer oftransduced cells. Cell proliferation will be induced with exogenousIL-12 (100 units/ml) for 48 h. Cells are counted and then re-plated atdensities of 5×10⁵ or 1×10⁶ cells per ml in a 12-well dish with 1 mlcomplete culture medium per well. Virus and ConA are added together atthe time of re-plating in the presence of polybrene (8 μg/ml). Themedium is changed 24 hours after infection with complete mediumcontaining 100 units of recombinant IL-2 per ml. Aliquots of thetransduced cells are examined, for Fas-ligand expression, at 48 hoursafter infection via flow cytometry.

Animals will receive standardized numbers of cytokine-producing cells orcontrol mock-transfected cells intraperitoneally. Concentrated cellsuspensions are injected directly into the mouse synovium, as describedin section 4A above. In parallel, aliquots of the transferred cellpopulations are maintained in tissue culture supplemented with exogenousIL-2.

Mice are monitored in a blinded fashion for signs of arthritis. The dateof disease onset is recorded and clinical severity of each joint orgroup of joints (toes, tarsus, ankle, wrist, knee) are graded asfollows: 0 (normal), 1 (erythema), 2 (swelling), 3 (deformity), 4(necrosis). The scores are summed to yield the arthritic score. Theseverity of arthritis is expressed both as the mean score observed on agiven day, and as the mean of the maximal arthritic score reached byeach mouse during the clinical course of the disease. At the time ofdeath, hind paws are dissected free and processed for histologicexamination or for RT-PCR. The histologic severity of the arthritis isscored on a scale of 0-3 for synovial proliferation and inflammatorycell infiltration, where a score of 0=normal and 3=severe.

For mice receiving intra-synovial injection of control of testadenovirus vector, the level of arthritis observed between contralateralsites is compared. In addition, the overall joint score minus that ofthe injected joint for the entire animal is compared with that observedin the joint injected with the control or test adenovirus vector.

Local administration of Fas-ligand adenovirus expression vectors willresult in clearance of activated cells, as assessed by measuring therelative levels of CD80 mRNA by quantitative RT-PCR. This treatment alsowill lead to an enhanced level. Also, whether such level of apoptosisidentified in affected mouse synovial tissue is assessed by the TUNELassay (“Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP NickEnd Labeling”). TUNEL is performed by immersing the sections in TdTbuffer (30 mM Tris-HCl, pH 7.2, 140 nM sodium cacodylate, 1 mM cobaltchloride), and then adding TdT (GIBCO BRL, Grand Island, N.Y.) andbiotinylated dUTP (Boehringer Mannheim, Indianapolis, Ind.). Thereaction is terminated by immersing the sections in TB buffer (300 mMsodium chloride, 30 mM sodium citrate). Subsequently, the samples aretreated with peroxidase-labeled streptavidin and then visualized usingthe VECTASTAIN ABC kit (Vector Laboratories Inc., Burlingame, Calif.).For immunohistochemistry, the sections are blocked with 4% skim milk for30 minutes at room temperature, then incubated with biotinylated mAbsspecific for mouse CD3, B220, CD80, or CD95 (Fas). These antibodies areavailable from Pharmingen (San Diego, Calif.).

b. Treatment of Rheumatoid Arthritis Patients with a Gene Therapy VectorEncoding an Accessory Molecule Ligand, Fas Ligand

Candidate Fas-ligand constructs identified as having potentialtherapeutic benefit are used in human protocols to treat RA. Humanprotocols encompass either in vivo or ex vivo methods to deliver theFas-ligand constructs. Furthermore, the Fas-ligand constructs arepotentially delivered by either viral or non-viral methods. Outlines oftherapeutic strategies are described below.

An ex vivo therapy is similar to a protocol described forintra-articular transplantation of autologous synoviocytes retrovirallytransduced to synthesize interleukin-1 receptor antagonist (Evan,Christopher et. al., Clinical Trial to Assess the Safety, Feasibility,and Efficacy of Transferring a Potentially Anti-Arthritic Cytokine Geneto Human Joints with Rheumatoid arhtritis, Human Gene Therapy, Vol. 7,1261-1280). In this procedure, after clinical diagnosis of RA, thesynovium is harvested during total joint replacement. The synoviocytesre-isolated and expanded, then transduced or transfected withheterologous Fas-ligand into synoviocytes (via retrovirus, adenovirus,naked DNA, etc.). The gene-modified synoviocytes are then reinjectedinto the patient, who is monitored and tested for amelioration ofRA-associated symptoms, and for expression and function of theFas-ligand in modified synoviocytes.

In another ex vivo protocol, an allogeneic immortalized cell line thatstably expresses the heterologous Fas-ligand is administered to the RApatient. In this protocol, a stable immortalized cell line expressingFas-ligand (introduced by transfection of the gene into the cell bynonviral methods, such as electroporation), or by viral transduction ofthe gene into the cell) is constructed. The modified cell line isinjected into the patient, who is monitored and tested for ameliorationof RA associated symptoms, and for expression and function of thehFas-ligand in modified synoviocytes.

An in vivo based therapy will is similar in concept to the ameliorationof collagen-induced-arthritis using a murine Fas-ligand adenovirus genetherapy vector, described in Zhang, et al., J. Clin. Invest.100:1951-1957 (1997). In our use of such an approach, delivery of thehFas-ligand construct or chimeric ΔfasL directly to the joints of RApatients is performed using either viral or non-viral methods. In thisprocedure, the Fas-ligand construct (e.g. hFas-ligand adenovirus) isdirectly injected into the synovium. Patients are monitored and testedfor amelioration of RA-associated symptoms as well as biological testingfor expression and function of the hFas-ligand in modified synoviocytes.

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 40. A chimeric nucleic acid moleculecomprising a polynucleotide encoding a first TNF family ligand domainsegment and a polynucleotide encoding a second TNF family ligand domainsegment, wherein: (a) one of the domain segments encoded is a Domain IIIsegment lacking a matrix metalloproteinase (mmp) cleavage site; (b) thefirst and second TNF family ligands are different members selected fromthe group of TNF family ligands consisting of CD40, CD70, Fas, TRAIL,CD30, 4-1BBL, NGF, TNF alpha and TNF beta; and, (c) the chimeric nucleicacid molecule encodes a polypeptide having TNF binding activity.
 41. Thechimeric nucleic acid molecule according to claim 1, wherein the firstTNF family ligand domain segment is a Domain III segment lacking amatrix metalloproteinase cleavage site, and the second TNF family liganddomain segment is Domain IV domain segment that contains a binding sitefor a receptor to a TNF family ligand selected from the group of TNFfamily ligands consisting of CD40, CD70, Fas, TRAIL, CD30, 4-1BBL, NGF,TNF alpha and TNF beta.
 42. The chimeric nucleic acid molecule accordingto claim 2, wherein the first polynucleotide further encodes a Domain IIdomain segment from the same TNF family ligand as either the Domain IIIor Domain IV domain segments.
 43. The chimeric nucleic acid moleculeaccording to claim 2 or claim 3, wherein the first polynucleotidefurther encodes a Domain I domain segment from the same TNF familyligand as either the Domain III or Domain IV domain segments.
 44. Thechimeric nucleic acid molecule according to claim 1, comprising thenucleotide sequence of SEQ.ID. No. 19, wherein Domain III of themolecule is modified to delete an mmp cleavage site.
 45. The chimericnucleic acid molecule according to claim 1, wherein the firstpolynucleotide encodes a Domain III domain segment encoded by anucleotide sequence contained in, and selected from, the groupconsisting of SEQ.ID. Nos. 2, 9, 10, 13, 15, 16, 17 and 41, wherein themolecule is modified to delete the nucleotide sequence coding for a mmpcleavage site.
 46. The chimeric nucleic acid molecule according to claim1 or claim 6, wherein the second polynucleotide encodes a Domain IVdomain segment encoded by a nucleotide sequence contained in, andselected from, the group consisting of SEQ.ID. Nos. 2, 9, 10, 13, 15,16, 17 and
 41. 47. A chimeric nucleic acid molecule comprising thenucleotide sequence of SEQ.ID. No.
 19. 48. A chimeric nucleic acidmolecule comprising the nucleotide sequence of SEQ.ID. No.20.
 49. Achimeric nucleic acid molecule comprising a polynucleotide encoding ahuman TNF family ligand domain segment and a polynucleotide encoding anon-human TNF family ligand domain segment, wherein: (a) one of thedomain segments encoded is a Domain segment lacking a matrixmetalloproteinase cleavage site; (b) the human and non-human TNF familyligands are selected from the group of TNF family ligands consisting ofCD40, CD70, Fas, TRAIL, CD30, 4-1BBL, NGF, TNF alpha and TNF beta; and,(c) the chimeric nucleic acid molecule encodes a polypeptide having TNFbinding activity.
 50. The chimeric nucleic acid molecule according toclaim 10, wherein the first TNF family ligand domain segment is a DomainIII segment lacking a matrix metalloproteinase cleavage site, and thesecond TNF family ligand domain segment is Domain IV domain segment thatcontains a binding site for a receptor to a TNF family ligand selectedfrom the group of TNF family ligands consisting of CD40, CD70, Fas,TRAIL, CD30, 4-1BBL, NGF, TNF alpha and TNF beta.
 51. The chimericnucleic acid molecule according to claim 11, wherein the firstpolynucleotide further encodes a Domain II domain segment from the samespecies as either the Domain III or Domain IV domain segments.
 52. Thechimeric nucleic acid molecule according to claim 11 or claim 12,wherein the first polynucleotide further encodes a Domain I domainsegment from the same species as either the Domain III or Domain IVdomain segments.
 53. The chimeric nucleic acid molecule according toclaim 10, comprising the nucleotide sequence of SEQ.ID. No.
 20. 54. Thechimeric nucleic acid molecule according to claim 10, wherein the firstpolynucleotide encodes a Domain III domain segment encoded by anucleotide sequence contained in, and selected from, the groupconsisting of SEQ.ID. Nos. 1, 8, 11, 12,

, 21, 22, 24, 25, 31, 36 and 42, wherein the molecule is modified todelete the nucleotide sequence coding for a mmp cleavage site.
 55. Thechimeric nucleic acid molecule according to claim 10 or claim 15,wherein the second polynucleotide encodes a Domain IV domain segmentencoded by a nucleotide sequence contained in, and selected from, thegroup consisting of SEQ.ID. Nos. 2, 9, 10, 13, 15, 16, 17 and
 41. 56. Agenetic construct comprising the chimeric nucleic acid moleculeaccording to claim 1 or claim
 10. 57. A host cell comprising thechimeric nucleic acid molecule according to claim 1 or claim
 10. 58. Anexpression vector comprising the chimeric nucleic acid moleculeaccording to claim 1 or claim
 10. 59. A polypeptide encoded by thechimeric nucleic acid molecule according to claim
 1. 60. A polypeptideencoded by the chimeric nucleic acid molecule according to claim
 10. 61.A method for increasing the concentration of a ligand capable of bindingto a TNF family ligand receptor on the surface of a human cell,comprising introducing into the cell a chimeric nucleic acid moleculeaccording to claim 1, whereby the polypeptide encoded by said nucleicacid molecule has improved stability on the surface of the cells than anative TNF family ligand molecule.
 62. The method according to claim 22,wherein the cell is CD40+.
 63. A method for increasing the concentrationof a ligand capable of binding to a TNF family ligand receptor on thesurface of a human cell, comprising introducing into the cell a chimericnucleic acid molecule according to claim 10, whereby the polypeptideencoded by said nucleic acid molecule has improved stability on thesurface of the cells than a native TNF family ligand molecule.
 64. Themethod according to claim 24, wherein the cell is CD40+.
 65. The methodaccording to claim 22 or claim 24, wherein the human cell is in situwithin, or is taken from, a person with a neoplasia, and wherein furtherintroducing the chimeric nucleic acid molecule into the cell followed byin vivo expression of the polypeptide encoded by said nucleic acidmolecule has a therapeutic effect on the neoplasia.